Non-endogenous, constitutively activated human G protein-coupled receptors

ABSTRACT

The invention disclosed in this patent document relates to transmembrane receptors, more particularly to a human G protein-coupled receptor for which the endogenous ligand is unknown (“orphan GPCR receptors”), and most particularly to mutated (non-endogenous) versions of the human GPCRs for evidence of constitutive activity.

[0001] This patent application is a continuation-in-part of, and claimspriority from, U.S. Ser. No. 09/170,496, filed with the U.S. patent andTrademark Office on Oct. 13, 1998. This application also claims thebenefit of priority from the following provisional applications, allfiled via U.S. Express Mail with the U.S. patent and Trademark Office onthe indicated dates: U.S. Provisional No. 60/110,060, filed Nov. 27,1998; U.S. Provisional No. 60/120,416, filed Feb. 16, 1999; U.S.Provisional No. 60/121,852, filed Feb. 26, 1999 claiming benefit of U.S.Provisional No. 60/109,213, filed Nov. 20, 1998; U.S. Provisional No.60/123,944, filed Mar. 12, 1999; U.S. Provisional No. 60/123,945, filedMar. 12, 1999; U.S. Provisional No. 60/123,948, filed Mar. 12, 1999;U.S. Provisional No. 60/123,951, filed Mar. 12, 1999; U.S. ProvisionalNo. 60/123,946, filed Mar. 12, 1999; U.S. Provisional No. 60/123,949,filed Mar. 12, 1999; U.S. Provisional No. 60/152,524, filed Sep. 3,1999, claiming benefit of U.S. Provisional No. 60/151,114, filed Aug.27, 1999 and U.S. Provisional No. 60/108,029, filed Nov. 12, 1998; U.S.Provisional No. 60/136,436, filed May 28, 1999; U.S. Provisional No.60/136,439, filed May 28, 1999; U.S. Provisional No. 60/136,567, filedMay 28, 1999; U.S. Provisional No. 60/137,127, filed May 28, 1999; U.S.Provisional No. 60/137,131, filed May 28, 1999; U.S. Provisional No.60/141,448, filed Jun. 29, 1999 claiming benefit of U.S. Provisional No.60/136,437, filed May 28, 1999; U.S. Provisional No. 60/156,633, filedSep. 29, 1999; U.S. Provisional No. 60/156,555, filed Sep. 29, 1999;U.S. Provisional No. 60/156,634, filed Sep. 29, 1999;U.S. ProvisionalNo. ______ (Arena Pharmaceuticals, Inc. docket number: CHN10-1), filedSep. 29, 1999; U.S. Provisional No. ______ (Arena Pharmaceuticals, Inc.docket number: RUP6-1), filed Oct. 1, 1999; U.S. Provisional No. ______(Arena Pharmaceuticals, Inc. docket number: RUP7-1), filed Oct. 1, 1999;U.S. Provisional No. ______ (Arena Pharmaceuticals, Inc. docket number:CHN6-1), filed Oct. 1, 1999; U.S. Provisional No. ______ (ArenaPharmaceuticals, Inc. docket number: RUP5-1), filed Oct. 1, 1999; andU.S. Provisional No. ______ (Arena Pharmaceuticals, Inc. docket number:CHN9-1), filed Oct. 1, 1999. This application is also related toco-pending U.S. Ser. No. ______ (Woodcock, Washburn, Kurtz, Makiewicz &Norris, LLP docket number AREN-0050), filed on Oct. 12, 1999 (via U.S.Express Mail) and U.S. Ser. No. 09/364,425, filed on Jul. 30, 1999, bothincorporated herein by reference. Each of the foregoing applications areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

[0002] The invention disclosed in this patent document relates totransmembrane receptors, and more particularly to human Gprotein-coupled receptors, and specifically to GPCRs that have beenaltered to establish or enhance constitutive activity of the receptor.Preferably, the altered GPCRs are used for the direct identification ofcandidate compounds as receptor agonists, inverse agonists or partialagonists having potential applicability as therapeutic agents.

BACKGROUND OF THE INVENTION

[0003] Although a number of receptor classes exist in humans, by far themost abundant and therapeutically relevant is represented by the Gprotein-coupled receptor (GPCR or GPCRs) class. It is estimated thatthere are some 100,000 genes within the human genome, and of these,approximately 2%, or 2,000 genes, are estimated to code for GPCRs.Receptors, including GPCRs, for which the endogenous ligand has beenidentified are referred to as “known” receptors, while receptors forwhich the endogenous ligand has not been identified are referred to as“orphan” receptors. GPCRs represent an important area for thedevelopment of pharmaceutical products: from approximately 20 of the 100known GPCRs, 60% of all prescription pharmaceuticals have beendeveloped.

[0004] GPCRs share a common structural motif. All these receptors haveseven sequences of between 22 to 24 hydrophobic amino acids that formseven alpha helices, each of which spans the membrane (each span isidentified by number, i.e., transmembrane-1 (TM-1), transmebrane-2(TM-2), etc.). The transmembrane helices are joined by strands of aminoacids between transmembrane-2 and transmembrane-3, transmembrane-4 andtransmembrane-5, and transmembrane-6 and transmembrane-7 on theexterior, or “extracellular” side, of the cell membrane (these arereferred to as “extracellular” regions 1, 2 and 3 (EC-1, EC-2 and EC-3),respectively). The transmembrane helices are also joined by strands ofamino acids between transmembrane-1 and transmembrane-2, transmembrane-3and transmembrane-4, and transmembrane-5 and transmembrane-6 on theinterior, or “intracellular” side, of the cell membrane (these arereferred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3),respectively). The “carboxy” (“C”) terminus of the receptor lies in theintracellular space within the cell, and the “amino” (“N”) terminus ofthe receptor lies in the extracellular space outside of the cell.

[0005] Generally, when an endogenous ligand binds with the receptor(often referred to as “activation” of the receptor), there is a changein the conformation of the intracellular region that allows for couplingbetween the intracellular region and an intracellular “G-protein.” Ithas been reported that GPCRs are “promiscuous” with respect to Gproteins, i.e., that a GPCR can interact with more than one G protein.See, Kenakin, T., 43 Life Sciences 1095 (1988). Although other Gproteins exist, currently, Gq, Gs, Gi, Gz and Go are G proteins thathave been identified. Endogenous ligand-activated GPCR coupling with theG-protein begins a signaling cascade process (referred to as “signaltransduction”). Under normal conditions, signal transduction ultimatelyresults in cellular activation or cellular inhibition. It is thoughtthat the IC-3 loop as well as the carboxy terminus of the receptorinteract with the G protein.

[0006] Under physiological conditions, GPCRs exist in the cell membranein equilibrium between two different conformations: an “inactive” stateand an “active” state. A receptor in an inactive state is unable to linkto the intracellular signaling transduction pathway to produce abiological response. Changing the receptor conformation to the activestate allows linkage to the transduction pathway (via the G-protein) andproduces abiological response.

[0007] A receptor may be stabilized in an active state by an endogenousligand or a compound such as a drug. Recent discoveries, including butnot exclusively limited to modifications to the amino acid sequence ofthe receptor, provide means other than endogenous ligands or drugs topromote and stabilize the receptor in the active state conformation.These means effectively stabilize the receptor in an active state bysimulating the effect of an endogenous ligand binding to the receptor.Stabilization by such ligand-independent means is termed “constitutivereceptor activation.”

SUMMARY OF THE INVENTION

[0008] Disclosed herein are non-endogenous versions of endogenous, humanGPCRs and uses thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 is a representation of 8XCRE-Luc reporter plasmid (see,Example 4(c)3.)

[0010]FIGS. 2A and 2B are graphic representations of the results of ATPand ADP binding to endogenous TDAG8 (2A) and comparisons in serum andserum free media (2B).

[0011]FIG. 3 is a graphic representation of the comparative signalingresults of CMV versus the GPCR Fusion Protein H9(F236K):Gsa.

DETAILED DESCRIPTION

[0012] The scientific literature that has evolved around receptors hasadopted a number of terms to refer to ligands having various effects onreceptors. For clarity and consistency, the following definitions willbe used throughout this patent document. To the extent that thesedefinitions conflict with other definitions for these terms, thefollowing definitions shall control:

[0013] AGONISTS shall mean materials (e.g., ligands, candidatecompounds) that activate the intracellular response when they bind tothe receptor, or enhance GTP binding to membranes. TABLE A AMINO ACIDABBREVIATIONS used herein are set out in Table A: ALANINE ALA A ARGININEARG R ASPARAGNE ASN N ASPARTIC ACID ASP D CYSTEINE CYS C GLUTAMIC ACIDGLU E GLUTAMINE GLN Q GLYCINE GLY G HISTIDINE HIS H ISOLEUCINE ILE ILEUCINE LEU L LYSINE LYS K METHIONINE MET M PHENYLALANINE PHE F PROLINEPRO P SERINE SER S THREONINE THR T TRYPTOPHAN TRP W TYROSINE TYR YVALINE VAL V

[0014] PARTIAL AGONISTS shall mean materials (e.g., ligands, candidatecompounds) that activate the intracellular response when they bind tothe receptor to a lesser degree/extent than do agonists, or enhance GTPbinding to membranes to a lesser degree/extent than do agonists.

[0015] ANTAGONIST shall mean materials (e.g., ligands, candidatecompounds) that competitively bind to the receptor at the same site asthe agonists but which do not activate the intracellular responseinitiated by the active form of the receptor, and can thereby inhibitthe intracellular responses by agonists or partial agonists. ANTAGONISTSdo not diminish the baseline intracellular response in the absence of anagonist or partial agonist.

[0016] CANDIDATE COMPOUND shall mean a molecule (for example, and notlimitation, a chemical compound) that is amenable to a screeningtechnique. Preferably, the phrase “candidate compound” does not includecompounds which were publicly known to be compounds selected from thegroup consisting of inverse agonist, agonist or antagonist to areceptor, as previously determined by an indirect identification process(“indirectly identified compound”); more preferably, not including anindirectly identified compound which has previously been determined tohave therapeutic efficacy in at least one mammal; and, most preferably,not including an indirectly identified compound which has previouslybeen determined to have therapeutic utility in humans.

[0017] COMPOSITION means a material comprising at least one component; a“pharmaceutical composition” is an example of a composition.

[0018] COMPOUND EFFICACY shall mean a measurement of the ability of acompound to inhibit or stimulate receptor functionality, as opposed toreceptor binding affinity. Exemplary means of detecting compoundefficacy are disclosed in the Example section of this patent document.

[0019] CODON shall mean a grouping of three nucleotides (or equivalentsto nucleotides) which generally comprise a nucleoside (adenosine (A),guanosine (G), cytidine (C), uridine (U) and thymidine (T)) coupled to aphosphate group and which, when translated, encodes an amino acid.

[0020] CONSTITUTIVELY ACTIVATED RECEPTOR shall mean a receptor subjectto constitutive receptor activation. A constitutively activated receptorcan be endogenous or non-endogenous.

[0021] CONSTITUTIVE RECEPTOR ACTIVATION shall mean stabilization of areceptor in the active state by means other than binding of the receptorwith its endogenous ligand or a chemical equivalent thereof.

[0022] CONTACT or CONTACTING shall mean bringing at least two moietiestogether, whether in an in vitro system or an in vivo system.

[0023] DIRECTLY IDENTIFYING or DIRECTLY IDENTIFIED, in relationship tothe phrase “candidate compound”, shall mean the screening of a candidatecompound against a constitutively activated receptor, preferably aconstitutively activated orphan receptor, and most preferably against aconstitutively activated G protein-coupled cell surface orphan receptor,and assessing the compound efficacy of such compound. This phrase is,under no circumstances, to be interpreted or understood to beencompassed by or to encompass the phrase “indirectly identifying” or“indirectly identified.”

[0024] ENDOGENOUS shall mean a material that a mammal naturallyproduces. ENDOGENOUS in reference to, for example and not limitation,the term “receptor,” shall mean that which is naturally produced by amammal (for example, and not limitation, a human) or a virus. Bycontrast, the term NON-ENDOGENOUS in this context shall mean that whichis not naturally produced by a mammal (for example, and not limitation,a human) or a virus. For example, and not limitation, a receptor whichis not constitutively active in its endogenous form, but whenmanipulated becomes constitutively active, is most preferably referredto herein as a “non-endogenous, constitutively activated receptor.” Bothterms can be utilized to describe both “in vivo” and “in vitro” systems.For example, and not limitation, in a screening approach, the endogenousor non-endogenous receptor may be in reference to an in vitro screeningsystem. As a further example and not limitation, where the genome of amammal has been manipulated to include a non-endogenous constitutivelyactivated receptor, screening of a candidate compound by means of an invivo system is viable.

[0025] G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSIONPROTEIN, in the context of the invention disclosed herein, each mean anon-endogenous protein comprising an endogenous, constitutively activateGPCR or a non-endogenous, constitutively activated GPCR fused to atleast one G protein, most preferably the alpha (a) subunit of such Gprotein (this being the subunit that binds GTP), with the G proteinpreferably being of the same type as the G protein that naturallycouples with endogenous orphan GPCR. For example, and not limitation, inan endogenous state, if the G protein “Gsa” is the predominate G proteinthat couples with the GPCR, a GPCR Fusion Protein based upon thespecific GPCR would be a non-endogenous protein comprising the GPCRfused to Gsa; in some circumstances, as will be set forth below, anon-predominant G protein can be fused to the GPCR. The G protein can befused directly to the c-terminus of the constitutively active GPCR orthere may be spacers between the two.

[0026] HOST CELL shall mean a cell capable of having a Plasmid and/orVector incorporated therein. In the case of a prokaryotic Host Cell, aPlasmid is typically replicated as a autonomous molecule as the HostCell replicates (generally, the Plasmid is thereafter isolated forintroduction into a eukaryotic Host Cell); in the case of a eukaryoticHost Cell, a Plasmid is integrated into the cellular DNA of the HostCell such that when the eukaryotic Host Cell replicates, the Plasmidreplicates. Preferably, for the purposes of the invention disclosedherein, the Host Cell is eukaryotic, more preferably, mammalian, andmost preferably selected from the group consisting of 293, 293T andCOS-7 cells.

[0027] INDIRECTLY IDENTIFYING or INDIRECTLY IDENTIFIED means thetraditional approach to the drug discovery process involvingidentification of an endogenous ligand specific for an endogenousreceptor, screening of candidate compounds against the receptor fordetermination of those which interfere and/or compete with theligand-receptor interaction, and assessing the efficacy of the compoundfor affecting at least one second messenger pathway associated with theactivated receptor.

[0028] INHIBIT or INHIBITING, in relationship to the term “response”shall mean that a response is decreased or prevented in the presence ofa compound as opposed to in the absence of the compound.

[0029] INVERSE AGONISTS shall mean materials (e.g., ligand, candidatecompound) which bind to either the endogenous form of the receptor or tothe constitutively activated form of the receptor, and which inhibit thebaseline intracellular response initiated by the active form of thereceptor below the normal base level of activity which is observed inthe absence of agonists or partial agonists, or decrease GTP binding tomembranes. Preferably, the baseline intracellular response is inhibitedin the presence of the inverse agonist by at least 30%, more preferablyby at least 50%, and most preferably by at least 75%, as compared withthe baseline response in the absence of the inverse agonist.

[0030] KNOWN RECEPTOR shall mean an endogenous receptor for which theendogenous ligand specific for that receptor has been identified.

[0031] LIGAND shall mean an endogenous, naturally occurring moleculespecific for an endogenous, naturally occurring receptor.

[0032] MUTANT or MUTATION in reference to an endogenous receptor'snucleic acid and/or amino acid sequence shall mean a specified change orchanges to such endogenous sequences such that a mutated form of anendogenous, non-constitutively activated receptor evidences constitutiveactivation of the receptor. In terms of equivalents to specificsequences, a subsequent mutated form of a human receptor is consideredto be equivalent to a first mutation of the human receptor if (a) thelevel of constitutive activation of the subsequent mutated form of ahuman receptor is substantially the same as that evidenced by the firstmutation of the receptor; and (b) the percent sequence (amino acidand/or nucleic acid) homology between the subsequent mutated form of thereceptor and the first mutation of the receptor is at least about 80%,more preferably at least about 90% and most preferably at least 95%.Ideally, and owing to the fact that the most preferred cassettesdisclosed herein for achieving constitutive activation includes a singleamino acid and/or codon change between the endogenous and thenon-endogenous forms of the GPCR, the percent sequence homology shouldbe at least 98%.

[0033] NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurringmolecule specific for an endogenous naturally occurring ligand whereinthe binding of a ligand to a receptor activates an intracellularsignaling pathway.

[0034] ORPHAN RECEPTOR shall mean an endogenous receptor for which theendogenous ligand specific for that receptor has not been identified oris not known.

[0035] PHARMACEUTICAL COMPOSITION shall mean a composition comprising atleast one active ingredient, whereby the composition is amenable toinvestigation for a specified, efficacious outcome in a mammal (forexample, and not limitation, a human). Those of ordinary skill in theart will understand and appreciate the techniques appropriate fordetermining whether an active ingredient has a desired efficaciousoutcome based upon the needs of the artisan.

[0036] PLASMID shall mean the combination of a Vector and cDNA.Generally, a Plasmid is introduced into a Host Cell for the purposes ofreplication and/or expression of the cDNA as a protein.

[0037] STIMULATE or STIMULATING, in relationship to the term “response”shall mean that a response is increased in the presence of a compound asopposed to in the absence of the compound.

[0038] VECTOR in reference to cDNA shall mean a circular DNA capable ofincorporating at least one cDNA and capable of incorporation into a HostCell.

[0039] The order of the following sections is set forth forpresentational efficiency and is not intended, nor should be construed,as a limitation on the disclosure or the claims to follow.

[0040] A. Introduction

[0041] The traditional study of receptors has always proceeded from thea priori assumption (historically based) that the endogenous ligand mustfirst be identified before discovery could proceed to find antagonistsand other molecules that could affect the receptor. Even in cases wherean antagonist might have been known first, the search immediatelyextended to looking for the endogenous ligand. This mode of thinking haspersisted in receptor research even after the discovery ofconstitutively activated receptors. What has not been heretoforerecognized is that it is the active state of the receptor that is mostuseful for discovering agonists, partial agonists, and inverse agonistsof the receptor. For those diseases which result from an overly activereceptor or an under-active receptor, what is desired in a therapeuticdrug is a compound which acts to diminish the active state of a receptoror enhance the activity of the receptor, respectively, not necessarily adrug which is an antagonist to the endogenous ligand. This is because acompound that reduces or enhances the activity of the active receptorstate need not bind at the same site as the endogenous ligand. Thus, astaught by a method of this invention, any search for therapeuticcompounds should start by screening compounds against theligand-independent active state.

[0042] B. Identification of Human GPCRs

[0043] The efforts of the Human Genome project has led to theidentification of a plethora of information regarding nucleic acidsequences located within the human genome; it has been the case in thisendeavor that genetic sequence information has been made availablewithout an understanding or recognition as to whether or not anyparticular genomic sequence does or may contain open-reading frameinformation that translate human proteins. Several methods ofidentifying nucleic acid sequences within the human genome are withinthe purview of those having ordinary skill in the art. For example, andnot limitation, a variety of human GPCRs, disclosed herein, werediscovered by reviewing the GenBank™ database, while other GPCRs werediscovered by utilizing a nucleic acid sequence of a GPCR, previouslysequenced, to conduct a BLAST™ search of the EST database. Table B,below, lists several endogenous GPCRs that we have discovered, alongwith a GPCR's respective homologous receptor. TABLE B Disclosed Open PerCent Reference To Human Accession Reading Homology Homologous OrphanNumber Frame To Designated GPCR GPCRs Identified (Base Pairs) GPCR(Accession No.) hARE-3 AL033379 1,260 bp 52.3% LPA-R U92642 hARE-4AC006087 1,119 bp 36% P2Y5 AF000546 hARE-5 AC006255 1,104 bp 32% OryziasD43633 latipes hGPR27 AA775870 1,128 bp hARE-1 A1090920   999 bp 43%D13626 KIAA0001 hARE-2 AA359504 1,122 bp 53% GPR27 hPPR1 1167224 1,053bp 39% EBI1 L31581 hG2A AA754702 1,113 bp 31% GPR4 L36148 hRUP3 AL0354231,005 bp 30% 2133653 Drosophila melanogaster hRUP4 A1307658 1,296 bp 32%pNPGPR NP_004876 28% and 29% AAC41276 Zebra fish Ya and and Yb, AAB94616respectively hRUP5 AC005849 1,413 bp 25% DEZ Q99788 23% FMLPR P21462hRUP6 AC005871 1,245 bp 48% GPR66 NP_006047 hRUP7 AC007922 1,173 bp 43%H3R AF140538 hCHN3 EST 36581 1,113 bp 53% GPR27 hCHN4 AA804531 1,077 bp32% thrombin 4503637 hCHN6 EST 2134670 1,503 bp 36% edg-1 NP_001391hCHN8 EST 764455 1,029 bp 47% D13626 KIAA0001 hCHN9 EST 1541536 1,077 bp41% LTB4R NM_000752 hCHN10 EST 1365839 1,055bp 35% P2Y NM_002563

[0044] Receptor homology is useful in terms of gaining an appreciationof a role of the receptors within the human body. As the patent documentprogresses, we will disclose techniques for mutating these receptors toestablish non-endogenous, constitutively activated versions of thesereceptors.

[0045] The techniques disclosed herein have also been applied to otherhuman, orphan GPCRs known to the art, as will be apparent as the patentdocument progresses.

[0046] C. Receptor Screening

[0047] Screening candidate compounds against a non-endogenous,constitutively activated version of the human GPCRs disclosed hereinallows for the direct identification of candidate compounds which act atthis cell surface receptor, without requiring use of the receptor'sendogenous ligand. By determining areas within the body where theendogenous version of human GPCRs disclosed herein is expressed and/orover-expressed, it is possible to determine related disease/disorderstates which are associated with the expression and/or over-expressionof the receptor; such an approach is disclosed in this patent document.

[0048] With respect to creation of a mutation that may evidenceconstitutive activation of the human GPCR disclosed herein is based uponthe distance from the proline residue at which is presumed to be locatedwithin TM6 of the GPCR; this algorithmic technique is disclosed inco-pending and commonly assigned patent document U.S. Ser. No.09/170,496, incorporated herein by reference. The algorithmic techniqueis not predicated upon traditional sequence “alignment” but rather aspecified distance from the aforementioned TM6 proline residue. Bymutating the amino acid residue located 16 amino acid residues from thisresidue (presumably located in the IC3 region of the receptor) to, mostpreferably, a lysine residue, such activation may be obtained. Otheramino acid residues may be useful in the mutation at this position toachieve this objective.

[0049] D. Disease/Disorder Identification and/or Selection

[0050] As will be set forth in greater detail below, most preferablyinverse agonists to the non-endogenous, constitutively activated GPCRcan be identified by the methodologies of this invention. Such inverseagonists are ideal candidates as lead compounds in drug discoveryprograms for treating diseases related to this receptor. Because of theability to directly identify inverse agonists to the GPCR, therebyallowing for the development of pharmaceutical compositions, a searchfor diseases and disorders associated with the GPCR is relevant. Forexample, scanning both diseased and normal tissue samples for thepresence of the GPCR now becomes more than an academic exercise or onewhich might be pursued along the path of identifying an endogenousligand to the specific GPCR. Tissue scans can be conducted across abroad range of healthy and diseased tissues. Such tissue scans provide apreferred first step in associating a specific receptor with a diseaseand/or disorder. See, for example, co-pending application (docket numberARE-0050) for exemplary dot-blot and RT-PCR results of several of theGPCRs disclosed herein.

[0051] Preferably, the DNA sequence of the human GPCR is used to make aprobe for (a) dot-blot analysis against tissue-mRNA, and/or (b) RT-PCRidentification of the expression of the receptor in tissue samples. Thepresence of a receptor in a tissue source, or a diseased tissue, or thepresence of the receptor at elevated concentrations in diseased tissuecompared to a normal tissue, can be preferably utilized to identify acorrelation with a treatment regimen, including but not limited to, adisease associated with that disease. Receptors can equally well belocalized to regions of organs by this technique. Based on the knownfunctions of the specific tissues to which the receptor is localized,the putative functional role of the receptor can be deduced.

[0052] E. Screening of Candidate Compounds

[0053] 1. Generic GPCR screening assay techniques

[0054] When a G protein receptor becomes constitutively active, it bindsto a G protein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the binding ofGTP to the G protein. The G protein then acts as a GTPase and slowlyhydrolyzes the GTP to GDP, whereby the receptor, under normalconditions, becomes deactivated. However, constitutively activatedreceptors continue to exchange GDP to GTP. A non-hydrolyzable analog ofGTP, [³⁵S]GTPγS, can be used to monitor enhanced binding to membraneswhich express constitutively activated receptors. It is reported that[³⁵S]GTPγS can be used to monitor G protein coupling to membranes in theabsence and presence of ligand. An example of this monitoring, amongother examples well-known and available to those in the art, wasreported by Traynor and Nahorski in 1995. The preferred use of thisassay system is for initial screening of candidate compounds because thesystem is generically applicable to all G protein-coupled receptorsregardless of the particular G protein that interacts with theintracellular domain of the receptor.

[0055] 2. Specific GPCR Screening Assay Techniques

[0056] Once candidate compounds are identified using the “generic” Gprotein-coupled receptor assay (i.e., an assay to select compounds thatare agonists, partial agonists, or inverse agonists), further screeningto confirm that the compounds have interacted at the receptor site ispreferred. For example, a compound identified by the “generic” assay maynot bind to the receptor, but may instead merely “uncouple” the Gprotein from the intracellular domain.

[0057] a. Gs, Gz and Gi.

[0058] Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), onthe other hand, inhibit this enzyme. Adenylyl cyclase catalyzes theconversion of ATP to cAMP; thus, constitutively activated GPCRs thatcouple the Gs protein are associated with increased cellular levels ofcAMP. On the other hand, constitutively activated GPCRs that couple Gi(or Gz, Go) protein are associated with decreased cellular levels ofcAMP. See, generally, “Indirect Mechanisms of Synaptic Transmission,”Chpt. 8, From Neuron To Brain (3^(rd) Ed.) Nichols, J. G. et al eds.Sinauer Associates, Inc. (1992). Thus, assays that detect cAMP can beutilized to determine if a candidate compound is, e.g., an inverseagonist to the receptor (i.e., such a compound would decrease the levelsof cAMP). A variety of approaches known in the art for measuring cAMPcan be utilized; a most preferred approach relies upon the use ofanti-cAMP antibodies in an ELISA-based format. Another type of assaythat can be utilized is a whole cell second messenger reporter systemassay. Promoters on genes drive the expression of the proteins that aparticular gene encodes. Cyclic AMP drives gene expression by promotingthe binding of a cAMP-responsive DNA binding protein or transcriptionfactor (CREB) that then binds to the promoter at specific sites calledcAMP response elements and drives the expression of the gene. Reportersystems can be constructed which have a promoter containing multiplecAMP response elements before the reporter gene, e.g., β-galactosidaseor luciferase. Thus, a constitutively activated Gs-linked receptorcauses the accumulation of cAMP that then activates the gene andexpression of the reporter protein. The reporter protein such asβ-galactosidase or luciferase can then be detected using standardbiochemical assays (Chen et al. 1995).

[0059] b. Go and Gq.

[0060] Gq and Go are associated with activation of the enzymephospholipase C, which in turn hydrolyzes the phospholipid PIP2,releasing two intracellular messengers: diacycloglycerol (DAG) andinistol 1,4,5-triphoisphate (IP3). Increased accumulation of IP₃ isassociated with activation of Gq- and Go-associated receptors. See,generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, FromNeuron To Brain (3^(rd) Ed.) Nichols, J. G. et al eds. SinauerAssociates, Inc. (1992). Assays that detect IP3 accumulation can beutilized to determine if a candidate compound is, e.g., an inverseagonist to a Gq- or Go-associated receptor (i.e., such a compound woulddecrease the levels of IP₃). Gq-associated receptors can also beenexamined using an AP1 reporter assay in that Gq-dependent phospholipaseC causes activation of genes containing AP1 elements; thus, activatedGq-associated receptors will evidence an increase in the expression ofsuch genes, whereby inverse agonists thereto will evidence a decrease insuch expression, and agonists will evidence an increase in suchexpression. Commercially available assays for such detection areavailable.

[0061] 3. GPCR Fusion Protein

[0062] The use of an endogenous, constitutively activate orphan GPCR ora non-endogenous, constitutively activated orphan GPCR, for use inscreening of candidate compounds for the direct identification ofinverse agonists, agonists and partial agonists provide an interestingscreening challenge in that, by definition, the receptor is active evenin the absence of an endogenous ligand bound thereto. Thus, in order todifferentiate between, e.g., the non-endogenous receptor in the presenceof a candidate compound and the non-endogenous receptor in the absenceof that compound, with an aim of such a differentiation to allow for anunderstanding as to whether such compound may be an inverse agonist,agonist, partial agonist or have no affect on such a receptor, it ispreferred that an approach be utilized that can enhance suchdifferentiation. A preferred approach is the use of a GPCR FusionProtein.

[0063] Generally, once it is determined that a non-endogenous orphanGPCR has been constitutively activated using the assay techniques setforth above (as well as others), it is possible to determine thepredominant G protein that couples with the endogenous GPCR. Coupling ofthe G protein to the GPCR provides a signaling pathway that can beassessed. Because it is most preferred that screening take place by useof a mammalian expression system, such a system will be expected to haveendogenous G protein therein. Thus, by definition, in such a system, thenon-endogenous, constitutively activated orphan GPCR will continuouslysignal. In this regard, it is preferred that this signal be enhancedsuch that in the presence of, e.g., an inverse agonist to the receptor,it is more likely that it will be able to more readily differentiate,particularly in the context of screening, between the receptor when itis contacted with the inverse agonist.

[0064] The GPCR Fusion Protein is intended to enhance the efficacy of Gprotein coupling with the non-endogenous GPCR. The GPCR Fusion Proteinis preferred for screening with a non-endogenous, constitutivelyactivated GPCR because such an approach increases the signal that ismost preferably utilized in such screening techniques. This is importantin facilitating a significant “signal to noise” ratio; such asignificant ratio is import preferred for the screening of candidatecompounds as disclosed herein.

[0065] The construction of a construct useful for expression of a GPCRFusion Protein is within the purview of those having ordinary skill inthe art. Commercially available expression vectors and systems offer avariety of approaches that can fit the particular needs of aninvestigator. The criteria of importance for such a GPCR Fusion Proteinconstruct is that the endogenous GPCR sequence and the G proteinsequence both be in-frame (preferably, the sequence for the endogenousGPCR is upstream of the G protein sequence) and that the “stop” codon ofthe GPCR must be deleted or replaced such that upon expression of theGPCR, the G protein can also be expressed. The GPCR can be linkeddirectly to the G protein, or there can be spacer residues between thetwo (preferably, no more than about 12,although this number can bereadily ascertained by one of ordinary skill in the art). We have apreference (based upon convenience) of use of a spacer in that somerestriction sites that are not used will, effectively, upon expression,become a spacer. Most preferably, the G protein that couples to thenon-endogenous GPCR will have been identified prior to the creation ofthe GPCR Fusion Protein construct. Because there are only a few Gproteins that have been identified, it is preferred that a constructcomprising the sequence of the G protein (i.e., a universal G proteinconstruct) be available for insertion of an endogenous GPCR sequencetherein; this provides for efficiency in the context of large-scalescreening of a variety of different endogenous GPCRs having differentsequences.

[0066] As noted above, constitutively activated GPCRs that couple to Gi,Gz and Go are expected to inhibit the formation of cAMP making assaysbased upon these types of GPCRs challenging (i.e., the cAMP signaldecreases upon activation thus making the direct identification of, e.g,inverse agonists (which would further decrease this signal),interesting). As will be disclosed herein, we have ascertained that forthese types of receptors, it is possible to create a GPCR Fusion Proteinthat is not based upon the endogenous GPCR's endogenous G protein, in aneffort to establish a viable cyclase-based assay. Thus, for example, aGz coupled receptor such as H9, a GPCR Fusion Protein can be establishedthat utilizes a Gs fusion protein—we believe that such a fusionconstruct, upon expression, “drives” or “forces” the non-endogenous GPCRto couple with, e.g., Gs rather than the “natural” Gz protein, such thata cyclase-based assay can be established. Thus, for Gi, Gz and Gocoupled receptors, we prefer that that when a GPCR Fusion Protein isused and the assay is based upon detection of adenyl cyclase activity,that the fusion construct be established with Gs (or an equivalent Gprotein that stimulates the formation of the enzyme adenylyl cyclase).

[0067] F. Medicinal Chemistry

[0068] Generally, but not always, direct identification of candidatecompounds is preferably conducted in conjunction with compoundsgenerated via combinatorial chemistry techniques, whereby thousands ofcompounds are randomly prepared for such analysis. Generally, theresults of such screening will be compounds having unique corestructures; thereafter, these compounds are preferably subjected toadditional chemical modification around a preferred core structure(s) tofurther enhance the medicinal properties thereof. Such techniques areknown to those in the art and will not be addressed in detail in thispatent document.

[0069] G. Pharmaceutical Compositions

[0070] Candidate compounds selected for further development can beformulated into pharmaceutical compositions using techniques well knownto those in the art. Suitable pharmaceutically-acceptable carriers areavailable to those in the art; for example, see Remington'sPharmaceutical Sciences, 16^(th) Edition, 1980, Mack Publishing Co.,(Oslo et al., eds.)

[0071] H. Other Utility

[0072] Although a preferred use of the non-endogenous versions the humanGPCRs disclosed herein may be for the direct identification of candidatecompounds as inverse agonists, agonists or partial agonists (preferablyfor use as pharmaceutical agents), these versions of human GPCRs canalso be utilized in research settings. For example, in vitro and in vivosystems incorporating GPCRs can be utilized to further elucidate andunderstand the roles these receptors play in the human condition, bothnormal and diseased, as well as understanding the role of constitutiveactivation as it applies to understanding the signaling cascade. Thevalue in non-endogenous human GPCRs is that their utility as a researchtool is enhanced in that, because of their unique features,non-endogenous human GPCRs can be used to understand the role of thesereceptors in the human body before the endogenous ligand therefor isidentified. Other uses of the disclosed receptors will become apparentto those in the art based upon, inter alia, a review of this patentdocument.

EXAMPLES

[0073] The following examples are presented for purposes of elucidation,and not limitation, of the present invention. While specific nucleicacid and amino acid sequences are disclosed herein, those of ordinaryskill in the art are credited with the ability to make minormodifications to these sequences while achieving the same orsubstantially similar results reported below. The traditional approachto application or understanding of sequence cassettes from one sequenceto another (e.g. from rat receptor to human receptor or from humanreceptor A to human receptor B) is generally predicated upon sequencealignment techniques whereby the sequences are aligned in an effort todetermine areas of commonality. The mutational approach disclosed hereindoes not rely upon this approach but is instead based upon analgorithmic approach and a positional distance from a conserved prolineresidue located within the TM6 region of human GPCRs. Once this approachis secured, those in the art are credited with the ability to make minormodifications thereto to achieve substantially the same results (i.e.,constitutive activation) disclosed herein. Such modified approaches areconsidered within the purview of this disclosure

Example 1

[0074] Endogenous Human GPCRs

[0075] 1. Identification of Human GPCRs

[0076] Certain of the disclosed endogenous human GPCRs were identifiedbased upon a review of the GenBank™ database information. Whilesearching the database, the following cDNA clones were identified asevidenced below (Table C). TABLE C Disclosed Open Nucleic Amino HumanComplete DNA Reading Acid Acid Orphan Accession Sequence Frame SEQ. SEQ.GPCRs Number (Base Pairs) (Base Pairs) ID.NO. ID.NO. hARE-3 AL033379111,389 bp 1,260 bp 1 2 hARE-4 AC006087 226,925 bp 1,119 bp 3 4 hARE-5AC006255 127,605 bp 1,104 bp 5 6 hRUP3 AL035423 140,094 bp 1,005 bp 7 8hRUP5 AC005849 169,144 bp 1,413 bp 9 10 hRUP6 AC005871 218,807 bp 1,245bp 11 12 hRUP7 AC007922 158,858 bp 1,173 bp 13 14

[0077] Other disclosed endogenous human GPCRs were identified byconducting a BLAST™ search of EST database (dbest) using the followingEST clones as query sequences. The following EST clones identified werethen used as a probe to screen a human genomic library (Table D). TABLED Disclosed Open Human EST Clone/ Reading Orphan Query Accession No.Frame Nucleic Acid Amino Acid GPCRs (Sequence) Identified (Base Pairs)SEQ.ID.NO. SEQ.ID.NO. hGPCR27 Mouse AA775870 1,125 bp 17 18 GPCR27hARE-1 TDAG 1689643   999 bp 19 20 AI090920 hARE-2 GPCR27 68530 1,122 bp21 22 AA359504 hPPR1 Bovine 238667 1,053 bp 23 24 PPR1 H67224 hG2A MouseSee Example 2(a), 1,113 bp 25 26 1179426 below hCHN3 N.A. EST 365811,113 bp 27 28 (full length) hCHN4 TDAG 1184934 1,077 bp 29 30 AA804531hCHN6 N.A. EST 2134670 1,503 bp 31 32 (full length) hCHN8 KIAA0001 EST764455 1,029 bp 33 34 hCHN9 1365839 EST 1541536 1,077 bp 35 36 hCHN10Mouse EST Human 1365839 1,005 bp 37 38 1365839 hRUP4 N.A. AI307658 1,296bp 39 40

[0078] 2. Full Length Cloning

[0079] a. Human G2A

[0080] Mouse EST clone 1179426 was used to obtain a human genomic clonecontaining all but three amino acid G2A coding sequences. The 5′ of thiscoding sequence was obtained by using 5′ RACE, and the template for PCRwas Clontech's Human Spleen Marathon-Ready™ cDNA. The disclosed humanG2A was amplified by PCR using the G2A cDNA specific primers for thefirst and second round PCR as shown in SEQ.ID.NO.: 41 and SEQ.ID.NO.:42as follows: 5′-CTGTGTACAGCAGTTCGCAGAGTG-3′ (SEQ.ID.NO.:41; 1^(st) roundPCR) 5′-GAGTGCCAGGCAGAGCAGGTAGAC-3′ (SEQ.ID.NO.:42; second round PCR).

[0081] PCR was performed using Advantage GC Polymerase Kit (Clontech;manufacturing instructions will be followed), at 94° C. for 30 secfollowed by 5 cycles of 94° C. for 5 sec and 72° C. for 4 min; and 30cycles of 94° C. for 5 sec and 70° for 4 min. An approximate 1.3 Kb PCRfragment was purified from agarose gel, digested with Hind III and Xba Iand cloned into the expression vector pRC/CMV2 (Invitrogen). Thecloned-insert was sequenced using the T7 Sequenase™ kit (USB Amersham;manufacturer instructions followed) and the sequence was compared withthe presented sequence. Expression of the human G2A was detected byprobing an RNA dot blot (Clontech; manufacturer instructions followed)with the P³²-labeled fragment.

[0082] b. CHN9

[0083] Sequencing of the EST clone 1541536 showed CHN9 to be a partialcDNA clone having only an initiation codon; i.e., the termination codonwas missing. When CHN9 was used to blast against data base (nr), the 3′sequence of CHN9 was 100% homologous to the 5′ untranslated region ofthe leukotriene B4 receptor cDNA, which contained a termination codon inthe frame with CHN9 coding sequence. To determine whether the 5′untranslated region of LTB4R cDNA was the 3′ sequence of CHN9, PCR wasperformed using primers based upon the 5′ sequence flanking theinitiation codon found in CHN9 and the 3′ sequence around thetermination codon found in the LTB4R 5′ untranslated region. The 5′primer sequence utilized was as follows:5′-CCCGAATTCCTGCTTGCTCCCAGCTTGGCCC-3′ (SEQ.ID.NO.:43; sense) and5′-TGTGGATCCTGCTGTCAAAGGTCCCATTCCGG-3′ (SEQ.ID.NO.:44; antisense).

[0084] PCR was performed using thymus cDNA as a template and rTthpolymerase (Perkin Elmer) with the buffer system provided by themanufacturer, 0.25 uM of each primer, and 0.2 mM of each 4 nucleotides.The cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 minand 72° C. for 1 min and 10 sec. A 1.1 kb fragment consistent with thepredicted size was obtained from PCR. This PCR fragment was subclonedinto pCMV (see below) and sequenced (see, SEQ.ID.NO.: 35).

[0085] c. RUP4

[0086] The full length RUP4 was cloned by RT-PCR with human brain cDNA(Clontech) as templates: 5′-TCACAATGCTAGGTGTGGTC-3′ (SEQ.ID.NO.:45;sense) and 5′-TGCATAGACAATGGGATTACAG-3′ (SEQ.ID.NO.:46; antisense).

[0087] PCR was performed using TaqPlus Precision™ polymerase(Stratagene; manufacturing instructions followed) by the followingcycles: 94° C. for 2 min; 94° C. 30 sec; 55° C. for 30 sec, 72° C. for45 sec, and 72° C. for 10 min. Cycles 2 through 4 were repeated 30times.

[0088] The PCR products were separated on a 1% agarose gel and a 500 bpPCR fragment was isolated and cloned into the pCRII-TOPO™ vector(Invitrogen) and sequenced using the T7 DNA Sequenase™ kit (Amsham) andthe SP6/T7 primers (Stratagene). Sequence analysis revealed that the PCRfragment was indeed an alternatively spliced form of AI3 0765 8 having acontinuous open reading frame with similarity to other GPCRs. Thecompleted sequence of this PCR fragment was as follows:5′-TCACAATGCTAGGTGTGGTCTGGCTGGTGGCAGTCATCGTAGGATCACCCATGTGGCAC(SEQ.ID.NO.:47)GTGCAACAACTTGAGATCAAATATGACTTCCTATATGAAAAGGAACACATCTGCTGCTTAAGAGTGGACCAGCCCTGTGCACCAGAAGATCTACACCACCTTCATCCTTGTCATCCTCTTCCTCCTGCCTCTTATGGTGATGCTTATTCTGTACGTAAAATTGGTTATGAACTTTGGATAAAGAAAAGAGTTGGGGATGGTTCAGTGCTTCGAACTATTCATGGAAAAGAAATGTCCAAAATAGCCAGGAAGAAGAAACGAGCTGTCATTATGATGGTGACAGTGGTGGCTCTCTTTGCTGTGCTGGGCACCATTCCATGTTGTCCATATGATGATTGAATACAGTAATTTTGAAAAGGAATATGATGATGTCACAATCAAGATGATTTTTGCTATCGTGCAAATTATTGGATTTTCCAACTCCATCTGTAATCCCATTGTCTATGCA-3′

[0089] Based on the above sequence, two sense oligonucleotide primersets: 5′-CTGCTTAGAAGAGTGGACCAG-3′ (SEQ.ID.NO.:48; oligo 1),5′-CTGTGCACCAGAAGATCTACAC-3′ (SEQ.IDNO.:49; oligo 2)

[0090] and two antisense oligonucleotide primer sets:5′-CAAGGATGAAGGTGGTGTAGA-3′ (SEQ.ID.NO.:50; oligo 3)5′-GTGTAGATGTTCTGGTGCAGAGG-3′ (SEQ.ID.NO.:51; oligo 4)

[0091] were used for 3′- and 5′-RACE PCR with a human brainMarathon-Ready™ cDNA (Clontech, Cat# 7400-1) as template, according tomanufacture's instructions. DNA fragments generated by the RACE PCR werecloned into the pCRII-TOPO™ vector (Invitrogen) and sequenced using theSP6/T7 primers (Stratagene) and some internal primers. The 3′ RACEproduct contained a poly(A) tail and a completed open reading frameending at a TAA stop codon. The 5′ RACE product contained an incomplete5′ end; i.e., the ATG initiation codon was not present.

[0092] Based on the new 5′ sequence, oligo 3 and the following primer:

[0093] 5′-GCAATGCAGGTCATAGTGAGC -3′ (SEQ.ID.NO.: 52; oligo 5)

[0094] were used for the second round of 5′ race PCR and the PCRproducts were analyzed as above. A third round of 5′ race PCR wascarried out utilizing antisense primers:5′-TGGAGCATGGTGACGGGAATGCAGAAG-3′ (SEQ.ID.NO.:53; oligo 6) and5′-GTGATGAGCAGGTCACTGAGCGCCAAG-3′ (SEQ.ID.NO.:54; oligo7).

[0095] The sequence of the 5′ RACE PCR products revealed the presence ofthe initiation codon ATG, and further round of 5′ race PCR did notgenerate any more 5′ sequence. The completed 5′ sequence was confirmedby RT-PCR using sense primer

[0096] 5′-GCAATGCAGGCGCTTAACATTAC-3′ (SEQ.ID.NO.: 55; oligo 8)

[0097] and oligo 4 as primers and sequence analysis of the 650 bp PCRproduct generated from human brain and heart cDNA templates (Clontech,Cat# 7404-1). The completed 3′ sequence was confirmed by RT-PCR usingoligo 2 and the following antisense primer:

[0098] 5′-TTGGGTTACAATCTGAAGGGCA-3′ (SEQ.ID.NO.:56; oligo 9)

[0099] and sequence analysis of the 670 bp PCR product generated fromhuman brain and heart cDNA templates. (Clontech, Cat# 7404-1).

[0100] d. RUP5

[0101] The full length RUP5 was cloned by RT-PCR using a sense primerupstream from ATG, the initiation codon (SEQ.ID.NO.:57), and anantisense primer containing TCA as the stop codon (SEQ.ID.NO.:58), whichhad the following sequences: 5′-ACTCCGTGTCCAGCAGGACTCTG-3′(SEQ.ID.NO.:57) 5′-TGCGTGTTCCTGGACCCTCACGTG-3′ (SEQ.ID.NO.:58)

[0102] and human peripheral leukocyte cDNA (Clontech) as a template.Advantage™ cDNA polymerase (Clontech) was used for the amplification ina 50 ul reaction by the following cycle with step 2 through step 4repeated 30 times: 94° C. for 30 sec; 94° for 15 sec; 69° for 40 sec;72° C. for 3 min; and 72° C. fro 6 min. A 1.4 kb PCR fragment wasisolated and cloned with the pCRII-TOPO™ vector (Invitrogen) andcompletely sequenced using the T7 DNA Sequenase™ kit (Amsham). See,SEQ.ID.NO.: 9.

[0103] e. RUP6

[0104] The full length RUP6 was cloned by RT-PCR using primers:5′-CAGGCCTTGGATTTTAATGTCAGGGATGG-3′ (SEQ.ID. NO.:59) and5′-GGAGAGTCAGCTCTGAAAGAATTCAGG-3′ (SEQ.ID. NO.:60);

[0105] and human thymus Marathon-Ready™ cDNA (Clontech) as a template.Advantage cDNA polymerase (Clontech, according to manufacturer'sinstructions) was used for the amplification in a 50 ul reaction by thefollowing cycle: 94° C. for 30 sec; 94° C. for 5 sec; 66° C. for 40 sec;72° C. for 2.5 sec and 72° C. for 7 min. Cycles 2 through 4 wererepeated 30 times. A 1.3 Kb PCR fragment was isolated and cloned intothe pCRII-TOPO™ vector (Invitrogen) and completely sequenced (see,SEQ.ID.NO.: 11) using the ABI Big Dye Terminator™ kit (P.E. Biosystem).

[0106] f. RUP7

[0107] The full length RUP7 was cloned by RT-PCR using primers:5′-TGATGTGATGCCAGATACTAATAGCAC-3′ (SEQ.ID.NO.:61; sense) and5′-CCTGATTCATTTAGGTGAGATTGAGAC-3′ (SEQ.ID.NO.:62; antisense)

[0108] and human peripheral leukocyte cDNA (Clontech) as a template.Advantage™ cDNA polymerase (Clontech) was used for the amplification ina 50 ul reaction by the following cycle with step 2 to step 4 repeated30 times: 94° C. for 2 minutes; 94° C. for 15 seconds; 60° C. for 20seconds; 72° C. for 2 minutes; 72° C. for 10 minutes. A 1.25 Kb PCRfragment was isolated and cloned into the pCRII-TOPO™ vector(Invitrogen) and completely sequenced using the ABI Big Dye Terminator™kit (P.E. Biosystem). See, SEQ.ID.NO.: 13.

[0109] 3. Angiotensin II Type 1 Receptor (“AT1”)

[0110] The endogenous human angiotensin II type 1 receptor (“AT1”) wasobtained by PCR using genomic DNA as template and rTth polymerase(Perkin Elmer) with the buffer system provided by the manufacturer, 0.25μM of each primer, and 0.2 mM of each 4 nucleotides. The cycle conditionwas 30 cycles of 94° C. for 1 min, 55° C. for 1 min and 72° C. for 1.5min. The 5′ PCR primer contains a HindIII site with the sequence:

[0111] 5′-CCCAAGCTTCCCCAGGTGTATTTGAT-3′ (SEQ.ID.NO.: 63)

[0112] and the 3′ primer contains a BamHI site with the followingsequence:

[0113] 5′-GTTGGATCCACATAATGCATTTCTC-3′ (SEQ.ID.NO.: 64).

[0114] The resulting 1.3 kb PCR fragment was digested with HindIII andBamHI and cloned into HindIII-BamHI site of pCMV expression vector. ThecDNA clone was fully sequenced. Nucleic acid (SEQ.ID.NO.: 65) and aminoacid (SEQ.ID.NO.: 66) sequences for human AT1 were thereafter determinedand verified.

[0115] 4. GPR38

[0116] To obtain GPR38, PCR was performed by combining two PCRfragments, using human genomic cDNA as template and rTth poymerase(Perkin Elmer) with the buffer system provided by the manufacturer, 0.25uM of each primer, and 0.2 mM of each 4 nucleotides. The cycle conditionfor each PCR reaction was 30 cycles of 94° C. for 1 min, 62° C. for 1min and 72° C. for 2 min.

[0117] The first fragment was amplified with the 5′ PCR primer thatcontained an end site with the following sequence:

[0118] 5′-ACCATGGGCAGCCCCTGGAACGGCAGC-3′ (SEQ.ID.NO.:67)

[0119] and a 3′ primer having the following sequence:

[0120] 5′-AGAACCACCACCAGCAGGACGCGGACGGTCTGCCGGTGG-3′ (SEQ.ID.NO.:68).

[0121] The second PCR fragment was amplified with a 5′ primer having thefollowing sequence:

[0122] 5′-GTCCGCGTCCTGCTGGTGGTGGTTCTGGCATTTATAATT-3′ (SEQ.ID.NO.: 69)

[0123] and a 3′ primer that contained a BamHI site and having thefollowing sequence:

[0124] 5′-CCTGGATCCTTATCCCATCGTCTTCACGTTAGC-3′ (SEQ.ID.NO.: 70).

[0125] The two fragments were used as templates to amplify GPR38, usingSEQ.ID.NO.: 67 and SEQ.ID.NO.: 70 as primers (using the above-notedcycle conditions). The resulting 1.44 kb PCR fragment was digested withBamHI and cloned into Blunt-BamHI site of pCMV expression vector.

[0126] 5. MC4

[0127] To obtain MC4, PCR was performed using human genomic cDNA astemplate and rTth poymerase (Perkin Elmer) with the buffer systemprovided by the manufacturer, 0.25 uM of each primer, and 0.2 mM of each4 nucleotides. The cycle condition for each PCR reaction was 30 cyclesof 94° C. for 1 min, 54° C. for 1 min and 72° C. for 1.5 min.

[0128] The 5′ PCR contained an EcoRI site with the sequence:

[0129] 5′-CTGGAATTCTCCTGCCAGCATGGTGA-3′ (SEQ.ID.NO.: 71)

[0130] and the 3′ primer contained a BamHI site with the sequence:

[0131] 5′-GCAGGATCCTATATTGCGTGCTCTGTCCCC′-3 (SEQ.ID.NO.: 72).

[0132] The 1.0 kb PCR fragment was digest with EcoRI and BamHI andcloned into EcoRI-BamHI site of pCMV expression vector. Nucleic acid(SEQ.ID.NO.: 73) and amino acid (SEQ.ID.NO.: 74) sequences for human MC4were thereafter determined.

[0133] 6. CCKB

[0134] To obtain CCKB, PCR was performed using human stomach cDNA astemplate and rTth poymerase (Perkin Elmer) with the buffer systemprovided by the manufacturer, 0.25 uM of each primer, and 0.2 mM of each4 nucleotides. The cycle condition for each PCR reaction was 30 cyclesof 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 30 sec.The 5′ PCR contained a HindIII site with the sequence:

[0135] 5′-CCGAAGCTTCGAGCTGAGTAAGGCGGCGGGCT-3′ (SEQ.ID.NO.: 75)

[0136] and the 3′ primer contained an EcoRI site with the sequence:

[0137] 5′-GTGGAATTCATTTGCCCTGCCTCAACCCCCA-3 (SEQ.ID.NO.: 76).

[0138] The resulting 1.44 kb PCR fragment was digest with HindIII andEcoRI and cloned into HindIII-EcoRI site of pCMV expression vector.Nucleic acid (SEQ.ID.NO.: 77) and amino acid (SEQ.ID.NO.: 78) sequencesfor human CCKB were thereafter determined.

[0139] 7. TDAG8

[0140] To obtain TDAG8, PCR was performed using genomic DNA as templateand rTth polymerase (Perkin Elmer) with the buffer system provided bythe manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 56°C. for 1 min and 72° C. for 1 min and 20 sec. The 5′ PCR primercontained a HindIII site with the following sequence:

[0141] 5′-TGCAAGCTTAAAAAGGAAAAAATGAACAGC-3′ (SEQ.ID.NO.: 79)

[0142] and the 3′ primer contained a BamHI site with the followingsequence:

[0143] 5′-TAAGGATCCCTTCCCTTCAAAACATCCTTG -3′ (SEQ.ID.NO.: 80).

[0144] The resulting 1.1 kb PCR fragment was digested with HindIII andBamHI and cloned into HindIII-BamHI site of pCMV expression vector.Three resulting clones sequenced contained three potential polymorphismsinvolving changes of amino acid 43 from Pro to Ala, amino acid 97 fromLys to Asn and amino acid 130 from Ile to Phe. Nucleic acid (SEQ.ID.NO.:81) and amino acid (SEQ.ID.NO.: 82) sequences for human TDAG8 werethereafter determined.

[0145] 8. H9

[0146] To obtain H9, PCR was performed using pituitary cDNA as templateand rTth polymerase (Perkin Elmer) with the buffer system provided bythe manufacturer, 0.25 μM of each primer, and 0.2 mM of each 4nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 62°C. for 1 min and 72° C. for 2 min. The 5′ PCR primer contained a HindIIIsite with the following sequence:

[0147] 5′-GGAAAGCTTAACGATCCCCAGGAGCAACAT-3′ (SEQ.ID.NO.:15)

[0148] and the 3′ primer contained a BamHI site with the followingsequence:

[0149] 5′-CTGGGATCCTACGAGAGCATTTTTCACACAG-3′ (SEQ.ID.NO.:16).

[0150] The resulting 1.9 kb PCR fragment was digested with HindIII andBamHI and cloned into HindIII-BamHI site of pCMV expression vector. H9contained three potential polymorphisms involving changes of amino acidP320S, S493N and amino acid G448A. Nucleic acid (SEQ.ID.NO.: 139) andamino acid (SEQ.ID.NO.: 140) sequences for human H9 were thereafterdetermined and verified.

Example 2

[0151] Preparation of Non-endogenous, Constitutively Activated GPCRs

[0152] Those skilled in the art are credited with the ability to selecttechniques for mutation of a nucleic acid sequence. Presented below areapproaches utilized to create non-endogenous versions of several of thehuman GPCRs disclosed above. The mutations disclosed below are basedupon an algorithmic approach whereby the 16^(th) amino acid (located inthe IC3 region of the GPCR) from a conserved proline residue (located inthe TM6 region of the GPCR, near the TM6/IC3 interface) is mutated, mostpreferably to a lysine amino acid residue.

[0153] 1. Tranformer Site-directed™ Mutagenesis

[0154] Preparation of non-endogenous human GPCRs may be accomplished onhuman GPCRs using Transformer Site-Directed™ Mutagenesis Kit (Clontech)according to the manufacturer instructions. Two mutagenesis primers areutilized, most preferably a lysine mutagenesis oligonucleotide thatcreates the lysine mutation, and a selection marker oligonucleotide. Forconvenience, the codon mutation to be incorporated into the human GPCRis also noted, in standard form (Table E): TABLE E Receptor IdentifierCodon Mutation hARE-3 F313K hARE-4 V233K hARE-5 A240K hGPCR14 L257KhGPCR27 C283K hARE-1 E232K hARE-2 G285K hPPR1 L239K hG2A K232A hRUP3L224K hRUP5 A236K hRUP6 N267K hRUP7 A302K hCHN4 V236K hMC4 A244K hCHN3S284K hCHN6 L352K hCHN8 N235K hCHN9 0223K hCHN10 L231K hH9 F236K

[0155] The following GPCRs were mutated according with the above methodusing the designated sequence primers (Table F). TABLE F LysineMutagenesis (SEQ. ID. NO.) Selection Marker Receptor Codon >5′-3′ orientation, mutation (SEQ. ID. NO.) Identifier Mutation sequenceunderlined 5′-3′ orientation hRUP4 V272K CAGGAAGAAGAAACGAGCCACTGTCACCATCATAATG TGTCATTATGATGGTGACA ACAGCTCGTTTCTTCTTCC GTG (83)TG(84) hAT1 see below alternative approach; see below alternativeapproach; see below hGPR38 V297K GGCCACCGGCAGACCAAAC CTCCTTCGGTCCTCCTATCGCGTCCTGCTG (85) GTTGTCAGAAGT (86) hCCKB V332K alternative approach; seebelow alternative approach; see below hTDAG8 I225K GGAAAAGAAGAGAATCAACTCCTTCGGTCCTCCTATC AAAACTACTTGTCAGCATC GTTGTCAGAAGT (88) (87) hH9 F236KGCTGAGGTTCGCAATAAAC CTCCTTCGGTCCTCCTATC TAACCATGTTTGTG (143)GTTGTCAGAAGT (144) hMC4 A244K GCCAATATGAAGGGAAAA CTCCTTCGGTCCTCCTATCATTACCTTGACCATC (137) GTTGTCAGAAAGT (138)

[0156] The non-endogenous human GPCRs were then sequenced and thederived and verified nucleic acid and amino acid sequences are listed inthe accompanying “Sequence Listing” appendix to this patent document, assummarized in Table G below: TABLE G Non Endogenous Human Nucleic AcidAmino Acid GPCR Sequence Listing Sequence Listing hRIJP4 SEQ.ID.NO.: 127SEQ.ID.NO.: 128 (V272K) hAT1 (see alternative approaches (seealternative approaches, (see alternative approaches below) below) below)hGPR38 SEQ.ID.NO.: 129 SEQ.ID.NO.: 130 (V297K) hCCKB SEQ.ID.NO.: 131SEQ.ID.NO.: 132 (V332K) HTDAG8 SEQ.ID.NO.: 133 SEQ.ID.NO.: 134 (1225K)hH9 SEQ.ID.NO.: 141 SEQ.ID.NO.: 142 (F236K) hMC4 SEQ.ID.NO.: 135SEQ.TD.NO.: 136 (A244K)

[0157] 2. Alternative Approaches for Creation of Non-endogenous HumanGPCRs

[0158] a. AT1

[0159] 1. F239K Mutation

[0160] Preparation of a non-endogenous, constitutively activated humanAT1 receptor was accomplished by creating an F239K mutation (see,SEQ.ID.NO.:89 for nucleic acid sequence, and SEQ.ID.NO.: 90 for aminoacid sequence). Mutagenesis was performed using TransformerSite-Directed Mutagenesis™ Kit (Clontech) according to the tomanufacturer's instructions. The two mutagenesis primers were used, alysine mutagenesis oligonucleotide (SEQ.ID.NO.: 91) and a selectionmarker oligonucleotide (SEQ.ID.NO.: 92), which had the followingsequences: 5′-CCAAGAAATGATGATATTAAAAAGATAATTATGGC-3′ (SEQ. ID. NO.: 91)5′-CTCCTTCGGTCCTCCTATCGTTGTCAGAAGT-3′ (SEQ. ID. NO.: 92),

[0161] respectively.

[0162] 2. N111A Mutation

[0163] Preparation of a non-endogenous human AT1 receptor was alsoaccomplished by creating an N111A mutation (see, SEQ.ID.NO.:93 fornucleic acid sequence, and SEQ.ID.NO.: 94 for amino acid sequence). TwoPCR reactions were performed using pfu polymerase (Stratagene) with thebuffer system provided by the manufacturer, supplemented with 10% DMSO,0.25 μM of each primer, and 0.5 mM of each 4 nucleotides. The 5′ PCRsense primer used had the following sequence:

[0164] 5′-CCCAAGCTTCCCCAGGTGTATTTGAT-3′ (SEQ.ID.NO.: 95)

[0165] and the antisense primer had the following sequence:

[0166] 5′-CCTGCAGGCGAAACTGACTCTGGCTGAAG-3′ (SEQ.ID.NO.: 96).

[0167] The resulting 400 bp PCR fragment was digested with HindIII siteand subcloned into HindIII-SmaI site of pCMV vector (5′ construct). The3′ PCR sense primer used had the following sequence:

[0168] 5′-CTGTACGCTAGTGTGTTTCTACTCACGTGTCTCAGCATTGAT-3′ (SEQ.ID.NO.: 97)

[0169] and the antisense primer had the following sequence:

[0170] 5′-GTTGGATCCACATAATGCATTTTCTC-3′ (SEQ.ID.NO.: 98)

[0171] The resulting 880 bp PCR fragment was digested with BamHI andinserted into Pst (blunted by T4 polymerase) and BamHI site of 5′construct to generated the full length N111A construct. The cyclecondition was 25 cycles of 94° C. for 1 min, 60° C. for 1 min and 72° C.for 1 min (5′ PCR) or 1.5 min (3′ PCR).

[0172] 3. AT2K255IC3 Mutation

[0173] Preparation of a non-endogenous, constitutively activated humanAT1 was accomplished by creating an AT2K255IC3 “domain swap” mutation(see, SEQ.ID.NO.:99 for nucleic acid sequence, and SEQ.ID.NO.: 100 foramino acid sequence). Restriction sites flanking IC3 of AT1 weregenerated to facilitate replacement of the IC3 with corresponding IC3from angiotensin II type 2 receptor (AT2). This was accomplished byperforming two PCR reactions. A 5′ PCR fragment (Fragment A) encodedfrom the 5′ untranslated region to the beginning of IC3 was generated byutilizing SEQ.ID.NO.: 63 as sense primer and the following sequence:

[0174] 5′-TCCGAATTCCAAAATAACTTGTAAGAATGATCAGAAA-3′ (SEQ. ID.NO.: 101)

[0175] as antisense primer. A 3′ PCR fragment (Fragment B) encoding fromthe end of IC3 to the 3′ untranslated region was generated by using thefollowing sequence:

[0176] 5′-AGATCTTAAGAAGATAATTATGGCAATTGTGCT-3′ (SEQ.ID.NO.: 102)

[0177] as sense primer and SEQ.ID.NO.: 64 as antisense primer. The PCRcondition was 30 cycles of 94° C. for 1 min, 55° C. for 1 min and 72° C.for 1.5 min using endogenous AT1 cDNA clone as template and pfupolymerase (Stratagene), with the buffer systems provided by themanufacturer, supplemented with 10% DMSO, 0.25 μM of each primer, and0.5 mM of each 4 nucleotides. Fragment A (720 bp) was digested withHindIII and EcoRI and subcloned. Fragment B was digested with BamHI andsubcloned into pCMV vector with an EcoRI site 5′ to the cloned PCRfragment.

[0178] The DNA fragment (Fragment C) encoding IC3 of AT2 with a L255Kmutation and containing an EcoRI cohesive end at 5′ and a AflII cohesiveend at 3′, was generated by annealing 2 synthetic oligonucleotideshaving the following sequences:5′AATTCGAAAACACTTACTGAAGACGAATAGCTATGGGAAGAACAGGAT AACCCGTGACCAAG-3′(sense; SEQ.ID.NO.: 103)5′TTAACTTGGTCACGGGTTATCCTGTTCTTCCCATAGCTATTCGTCTTC AGTAAGTGTTTTCG-3′(antisense; SEQ.ID.NO.: 104).

[0179] Fragment C was inserted in front of Fragment B through EcoRI andAflII site. The resulting clone was then ligated with the Fragment Athrough the EcoRI site to generate AT1 with AT2K255IC3.

[0180] 4. A243+ Mutation

[0181] Preparation of a non-endogenous human AT1 receptor was alsoaccomplished by creating an A243+ mutation (see, SEQ.ID.NO.: 105 fornucleic acid sequence, and SEQ.ID.NO.: 106 for amino acid sequence). AnA243+ mutation was constructed using the following PCR based strategy:Two PCR reactions was performed using pfu polymerase (Stratagene) withthe buffer system provided by the manufacturer supplemented with 10%DMSO, 0.25 μM of each primer, and 0.5 mM of each 4 nucleotides. The 5′PCR sense primer utilized had the following sequence:

[0182] 5′-CCCAAGCTTCCCCAGGTGTATTGAT-3′ (SEQ.ID.NO.:107)

[0183] and the antisense primer had the following sequence:

[0184] 5′-AAGCACAATTGCTGCATAATTATCTTAAAAATATCATC-3′ (SEQ.ID.NO.: 108).

[0185] The 3′ PCR sense primer utilized had the following sequence:

[0186] 5′-AAGATAATTATGGCAGCAATTGTGCTTCTTTTTCT=T-3′ (SEQ.ID.NO.: 109)

[0187] containing the Ala insertion and antisense primer:

[0188] 5′-GTTGGATCCACATAATGCATTTCTC-3′ (SEQ.ID.NO.: 110).

[0189] The cycle condition was 25 cycles of 94° C. for 1 min, 54° C. for1 min and 72° C. for 1.5 min. An aliquot of the 5′ and 3′ PCR were thenused as co-template to perform secondary PCR using the 5′ PCR senseprimer and 3′ PCR antisense primer. The PCR condition was the same asprimary PCR except the extention time was 2.5 min. The resulting PCRfragment was digested with HindIII and BamHI and subcloned into pCMVvector. (See, SEQ.ID.NO.: 105)

[0190] 4. CCKB

[0191] Preparation of the non-endogenous, constitutively activated humanCCKB receptor was accomplished by creating a V322K mutation (see,SEQ.ID.NO.: 111 for nucleic acid sequence and SEQ.ID.NO.: 112 for aminoacid sequence). Mutagenesis was performed by PCR via amplification usingthe wildtype CCKB from Example 1.

[0192] The first PCR fragment (1 kb) was amplified by using SEQ.ID.NO.:75 and an antisense primer comprising a V322K mutation:

[0193] 5′-CAGCAGCATGCGCTTCACGCGCTTCTTAGCCCAG-3′ (SEQ.ID.NO.: 113).

[0194] The second PCR fragment (0.44 kb) was amplified by using a senseprimer comprising the V322K mutation:

[0195] 5′-AGAAGCGCGTGAAGCGCATGCTGCTGGTGATCGTT-3′ (SEQ.ID.NO.:114) andSEQ.ID.NO.: 76.

[0196] The two resulting PCR fragments were then used as template foramplifying CCKB comprising V332K, using SEQ.ID.NO.: 75 and SEQ.ID.NO.:76 and the above-noted system and conditions. The resulting 1.44 kb PCRfragment containing the V332K mutation was digested with HindIII andEcoRI and cloned into HindIII-EcoRI site of pCMV expression vector.(See, SEQ.ID.NO.: 111).

[0197] 3. QuikChange™ Site-Directed™ Mutagenesis

[0198] Preparation of non-endogenous human GPCRs can also beaccomplished by using QuikChange™ Site-Directed™ Mutagenesis Kit(Stratagene, according to manufacturer's instructions). Endogenous GPCRis preferably used as a template and two mutagenesis primers utilized,as well as, most preferably, a lysine mutagenesis oligonucleotide and aselection marker oligonucleotide (included in kit). For convenience, thecodon mutation incorporated into the human GPCR and the respectiveoligonucleotides are noted, in standard form (Table H): TABLE H LysineMutagenesis (SEQ. ID. NO.) Selection Marker Receptor Codon5′-3′ orientation, mutation (SEQ. ID. NO.) Identifier Mutationunderlined 5′-3′ orientation hCHN3 S284K ATGGAGAAAAGAATCAAAAGAATATATAGAACATTCTTTT TGTTCTATATA (115) GATTCTTTTCTCCAT (116) hCHN6 L352KCGCTCTCTGGCCTTGAAGCGCAC GCTGAGCGTGCGCTTCA GCTCAGC (117) AGGCCAGAGAGCG(118) hCHN8 N235K CCCAGGAAAAAGGTGAAAGTCA GAAAACTTTGACTTTCAC AAGTTTTC(119) CTTTTTCCTGGG (120) hCHN9 G223K GGGGCGCGGGTGAAACGGCTGGGCTCACCAGCCGTTTCA TGAGC (121) CCCGCGCCCC (122) hCHN10 L231KCCCCTTGAAAAGCCTAAGAACTT GATGACCAAGTTCTTAG GGTCATC (123) GCTTTTCAAGGGG(124)

Example 3

[0199] Receptor Expression

[0200] Although a variety of cells are available to the art for theexpression of proteins, it is most preferred that mammalian cells beutilized. The primary reason for this is predicated upon practicalities,i.e., utilization of, e.g., yeast cells for the expression of a GPCR,while possible, introduces into the protocol a non-mammalian cell whichmay not (indeed, in the case of yeast, does not) include thereceptor-coupling, genetic-mechanism and secretary pathways that haveevolved for mammalian systems—thus, results obtained in non-mammaliancells, while of potential use, are not as preferred as that obtainedfrom mammalian cells. Of the mammalian cells, COS-7, 293 and 293T cellsare particularly preferred, although the specific mammalian cellutilized can be predicated upon the particular needs of the artisan.

[0201] On day one, 1×10⁷ 293T cells per 150 mm plate were plated out. Onday two, two reaction tubes were prepared (the proportions to follow foreach tube are per plate): tube A was prepared by mixing 20 μg DNA (e.g.,pCMV vector; pCMV vector with receptor cDNA, etc.) in 1.2 ml serum freeDMEM (Irvine Scientific, Irvine, Calif.); tube B was prepared by mixing120 μl lipofectamine (Gibco BRL) in 1.2 ml serum free DMEM. Tubes A andB were admixed by inversions (several times), followed by incubation atroom temperature for 30-45 min. The admixture is referred to as the“transfection mixture”. Plated 293T cells were washed with 1XPBS,followed by addition of 10 ml serum free DMEM. 2.4 ml of thetransfection mixture were added to the cells, followed by incubation for4 hrs at 37° C./5% CO₂. The transfection mixture was removed byaspiration, followed by the addition of 25 ml of DMEM/10% Fetal BovineSerum. Cells were incubated at 37° C./5% CO₂. After 72 hr incubation,cells were harvested and utilized for analysis.

Example 4

[0202] Assays for Determination of Constitutive Activity ofNon-endogenous GPCRs

[0203] A variety of approaches are available for assessment ofconstitutive activity of the non-endogenous human GPCRS. The followingare illustrative; those of ordinary skill in the art are credited withthe ability to determine those techniques that are preferentiallybeneficial for the needs of the artisan.

[0204] 1. Membrane Binding Assays: [³⁵S]GTPγS Assay

[0205] When a G protein-coupled receptor is in its active state, eitheras a result of ligand binding or constitutive activation, the receptorcouples to a G protein and stimulates the release of GDP and subsequentbinding of GTP to the G protein. The alpha subunit of the Gprotein-receptor complex acts as a GTPase and slowly hydrolyzes the GTPto GDP, at which point the receptor normally is deactivated.Constitutively activated receptors continue to exchange GDP for GTP. Thenon-hydrolyzable GTP analog, [³⁵S]GTPγS, can be utilized to demonstrateenhanced binding of [³⁵S]GTPγS to membranes expressing constitutivelyactivated receptors. The advantage of using [³⁵S]GTPγS binding tomeasure constitutive activation is that: (a) it is genericallyapplicable to all G protein-coupled receptors; (b) it is proximal at themembrane surface making it less likely to pick-up molecules which affectthe intracellular cascade.

[0206] The assay utilizes the ability of G protein coupled receptors tostimulate [³⁵S]GTPγS binding to membranes expressing the relevantreceptors. The assay can, therefore, be used in the directidentification method to screen candidate compounds to known, orphan andconstitutively activated G protein-coupled receptors. The assay isgeneric and has application to drug discovery at all G protein-coupledreceptors.

[0207] The [³⁵S]GTPγS assay can be incubated in 20 mM HEPES and between1 and about 20 mM MgCl₂ (this amount can be adjusted for optimization ofresults, although 20 mM is preferred) pH 7.4, binding buffer withbetween about 0.3 and about 1.2 nM [³⁵S]GTPγS (this amount can beadjusted for optimization of results, although 1.2 is preferred) and12.5 to 75 μg membrane protein (e.g, COS-7 cells expressing thereceptor; this amount can be adjusted for optimization, although 75 μgis preferred) and 1 μM GDP (this amount can be changed for optimization)for 1 hour. Wheatgerm agglutinin beads (25 μl; Amersham) should then beadded and the mixture incubated for another 30 minutes at roomtemperature. The tubes are then centrifuged at 1500× g for 5 minutes atroom temperature and then counted in a scintillation counter.

[0208] A less costly but equally applicable alternative has beenidentified which also meets the needs of large scale screening. Flashplates™ and Wallac™ scintistrips may be utilized to format a highthroughput [³⁵S]GTPγS binding assay. Furthermore, using this technique,the assay can be utilized for known GPCRs to simultaneously monitortritiated ligand binding to the receptor at the same time as monitoringthe efficacy via [³⁵S]GTPγS binding. This is possible because the Wallacbeta counter can switch energy windows to look at both tritium and³⁵S-labeled probes. This assay may also be used to detect other types ofmembrane activation events resulting in receptor activation. Forexample, the assay may be used to monitor ³²P phosphorylation of avariety of receptors (both G protein coupled and tyrosine kinasereceptors). When the membranes are centrifuged to the bottom of thewell, the bound [³⁵S]GTPγS or the ³²P-phosphorylated receptor willactivate the scintillant which is coated of the wells. Scinti® strips(Wallac) have been used to demonstrate this principle. In addition, theassay also has utility for measuring ligand binding to receptors usingradioactively labeled ligands. In a similar manner, when theradiolabeled bound ligand is centrifuged to the bottom of the well, thescintistrip label comes into proximity with the radiolabeled ligandresulting in activation and detection.

[0209] 2. Adenylyl Cyclase

[0210] A Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat.No. SMP004A) designed for cell-based assays can be modified for use withcrude plasma membranes. The Flash Plate wells contain a scintillantcoating which also contains a specific antibody recognizing cAMP. ThecAMP generated in the wells was quantitated by a direct competition forbinding of radioactive cAMP tracer to the cAMP antibody. The followingserves as a brief protocol for the measurement of changes in cAMP levelsin membranes that express the receptors.

[0211] Transfected cells are harvested approximately three days aftertransfection. Membranes were prepared by homogenization of suspendedcells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl₂.Homogenization is performed on ice using a Brinkman Polytron™ forapproximately 10 seconds. The resulting homogenate is centrifuged at49,000× g for 15 minutes at 4° C. The resulting pellet is thenresuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA,homogenized for 10 seconds, followed by centrifugation at 49,000× g for15 minutes at 4° C. The resulting pellet can be stored at −80° C. untilutilized. On the day of measurement, the membrane pellet is slowlythawed at room temperature, resuspended in buffer containing 20 mMHEPES, pH 7.4 and 10 mM MgCL₂ (these amounts can be optimized, althoughthe values listed herein are preferred), to yield a final proteinconcentration of 0.60 mg/ml (the resuspended membranes were placed onice until use). cAMP standards and Detection Buffer (comprising 2 μCi oftracer [¹²⁵I cAMP (100 μl] to 11 ml Detection Buffer) are prepared andmaintained in accordance with the manufacturer's instructions. AssayBuffer is prepared fresh for screening and contained 20 mM HEPES, pH7.4, 1 mM MgCl₂, 20 mM (Sigma), 0.1 units/ml creatine phosphokinase(Sigma), 50 ,uM GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer can bestored on ice until utilized. The assay is initiated by addition of 50ul of assay buffer followed by addition of 50 ul of membrane suspensionto the NEN Flash Plate. The resultant assay mixture is incubated for 60minutes at room temperature followed by addition of 100 ul of detectionbuffer. Plates are then incubated an additional 2-4 hours followed bycounting in a Wallac MicroBeta™ scintillation counter. Values ofcAMP/well are extrapolated from a standard cAMP curve that is containedwithin each assay plate.

[0212] C. Reporter-based Assays

[0213] 1. CREB Reporter Assay (Gs-associated Receptors)

[0214] A method to detect Gs stimulation depends on the known propertyof the transcription factor CREB, which is activated in a cAMP-dependentmanner. A PathDetect™ CREB trans-Reporting System (Stratagene, Catalogue#219010) can utilized to assay for Gs coupled activity in 293 or 293Tcells. Cells are transfected with the plasmids components of this abovesystem and the indicated expression plasmid encoding endogenous ormutant receptor using a Mammalian Transfection Kit (Stratagene,Catalogue #200285) according to the manufacturer's instructions.Briefly, 400 ng pFR-Luc (luciferase reporter plasmid containing Gal4recognition sequences), 40 ng pFA2-CREB (Gal4-CREB fusion proteincontaining the Gal4 DNA-binding domain), 80 ng pCMV-receptor expressionplasmid (comprising the receptor) and 20 ng CMV-SEAP (secreted alkalinephosphatase expression plasmid; alkaline phosphatase activity ismeasured in the media of transfected cells to control for variations intransfection efficiency between samples), are combined in a calciumphosphate precipitate as per the Kit's instructions. Half of theprecipitate is equally distributed over 3 wells in a 96-well plate, kepton the cells overnight, and replaced with fresh medium the followingmorning. Forty-eight (48) hr after the start of the transfection, cellsare treated and assayed for, e.g., luciferase activity

[0215] 2. AP1 Reporter Assay (Gq-associated Receptors)

[0216] A method to detect Gq stimulation depends on the known propertyof Gq-dependent phospholipase C to cause the activation of genescontaining API elements in their promoter. A Pathdetect™ AP-1cis-Reporting System (Stratagene, Catalogue #219073) can be utilizedfollowing the protocol set forth above with respect to the CREB reporterassay, except that the components of the calcium phosphate precipitatewere 410 ng pAP1-Luc, 80 ng pCMV-receptor expression plasmid, and 20 ngCMV-SEAP.

[0217] 3. CRE-LUC Reporter Assay

[0218] 293 and 293T cells are plated-out on 96 well plates at a densityof 2×10⁴ cells per well and were transfected using Lipofectamine Reagent(BRL) the following day according to manufacturer instructions. ADNA/lipid mixture is prepared for each 6-well transfection as follows:260 ng of plasmid DNA in 100 μl of DMEM were gently mixed with 2 μl oflipid in 100 μl of DMEM (the 260 ng of plasmid DNA consisted of 200 ngof a 8×CRE-Luc reporter plasmid (see below and FIG. 1 for arepresentation of a portion of the plasmid), 50 ng of pCMV comprisingendogenous receptor or non-endogenous receptor or pCMV alone, and 10 ngof a GPRS expression plasmid (GPRS in pcDNA3 (Invitrogen)). The8XCRE-Luc reporter plasmid was prepared as follows: vector SRIF-β-galwas obtained by cloning the rat somatostatin promoter (−71/+51) atBglV-HindIII site in the pβgal-Basic Vector (Clontech). Eight (8) copiesof cAMP response element were obtained by PCR from an adenovirustemplate AdpCF126CCRE8 (see, 7 Human Gene Therapy 1883 (1996)) andcloned into the SRIF-β-gal vector at the Kpn-BglV site, resulting in the8xCRE-β-gal reporter vector. The 8xCRE-Luc reporter plasmid wasgenerated by replacing the beta-galactosidase gene in the 8xCRE-β-galreporter vector with the luciferase gene obtained from the pGL3-basicvector (Promega) at the HindIII-BamHI site. Following 30 min. incubationat room temperature, the DNA/lipid mixture was diluted with 400 μl ofDMEM and 100 μl of the diluted mixture was added to each well. 100 μl ofDMEM with 10% FCS were added to each well after a 4 hr incubation in acell culture incubator. The following day the transfected cells werechanged with 200 μl/well of DMEM with 10% FCS. Eight (8) hours later,the wells were changed to 100 μl /well of DMEM without phenol red, afterone wash with PBS. Luciferase activity were measured the next day usingthe LucLite™ reporter gene assay kit (Packard) following manufacturerinstructions and read on a 1450 MicroBeta™ scintillation andluminescence counter (Wallac).

[0219] 4. SRF-Luc Reporter Assay

[0220] One method to detect Gq stimulation depends on the known propertyof Gq-dependent phospholipase C to cause the activation of genescontaining serum response factors in their promoter. A Pathdetect™SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gqcoupled activity in, e.g., COS7 cells. Cells are transfected with theplasmid components of the system and the indicated expression plasmidencoding endogenous or non-endogenous GPCR using a MammalianTransfection™ Kit (Stratagene, Catalogue #200285) according to themanufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ngpCMV-receptor expression plasmid and 20 ng CMV-SEAP (secreted alkalinephosphatase expression plasmid; alkaline phosphatase activity ismeasured in the media of transfected cells to control for variations intransfection efficiency between samples) are combined in a calciumphosphate precipitate as per the manufacturer's instructions. Half ofthe precipitate is equally distributed over 3 wells in a 96-well plate,kept on the cells in a serum free media for 24 hours. The last 5 hoursthe cells are incubated with 1 μM Angiotensin, where indicated. Cellsare then lysed and assayed for luciferase activity using a Luclite™ Kit(Packard, Cat. # 601691 1) and “Trilux 1450 Microbeta” liquidscintillation and luminescence counter (Wallac) as per themanufacturer's instructions. The data can be analyzed using GraphPadPrism™ 2.0a (GraphPad Software Inc.).

[0221] 5. Intracellular IP₃ Accumulation Assay

[0222] On day 1, cells comprising the receptors (endogenous and/ornon-endogenous) can be plated onto 24 well plates, usually 1×10⁵cells/well (although his umber can be optimized. On day 2 cells can betransfected by firstly mixing 0.25 ug DNA in 50 ul serum free DMEM/welland 2 ul lipofectamine in 50 μl serumfree DMEM/well. The solutions aregently mixed and incubated for 15-30 min at room temperature. Cells arewashed with 0.5 ml PBS and 400 ul of serum free media is mixed with thetransfection media and added to the cells. The cells are then incubatedfor 3-4 hrs at 37° C./5%CO₂ and then the transfection media is removedand replaced with 1 ml/well of regular growth media. On day 3 the cellsare labeled with ³H-myo-inositol. Briefly, the media is removed and thecells are washed with 0.5 ml PBS. Then 0.5 ml inositol-free/serum freemedia (GIBCO BRL) is added/well with 0.25 μCi of ³H-myo-inositol/welland the cells are incubated for 16-18 hrs o/n at 37° C./5%CO₂. On Day 4the cells are washed with 0.5 ml PBS and 0.45 ml of assay medium isadded containing inositol-free/serum free media 10 μM pargyline 10 mMlithium chloride or 0.4 ml of assay medium and 50 ul of 10× ketanserin(ket) to final concentration of 10 μM. The cells are then incubated for30 min at 37° C. The cells are then washed with 0.5 ml PBS and 200 ul offresh/icecold stop solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) isadded/well. The solution is kept on ice for 5-10 min or until cells werelysed and then neutralized by 200 μl of fresh/ice cold neutralizationsol. (7.5% HCL). The lysate is then transferred into 1.5 ml eppendorftubes and 1 ml of chloroform/methanol (1:2) is added/tube. The solutionis vortexed for 15 sec and the upper phase is applied to a BioradAG1-X8™ anion exchange resin (100-200 mesh). Firstly, the resin iswashed with water at 1:1.25 W/V and 0.9 ml of upper phase is loaded ontothe column. The column is washed with 10 mls of 5 mM myo-inositol and 10ml of 5 mM Na-borate/60 mM Na-formate. The inositol tris phosphates areeluted into scintillation vials containing 10 ml of scintillationcocktail with 2 ml of 0.1 M formic acid/ 1 M ammonium formate. Thecolumns are regenerated by washing with 10 ml of 0.1 M formic acid/3Mammonium formate and rinsed twice with dd H₂O and stored at 4° C. inwater.

[0223] Exemplary results are presented below in Table I: TABLE I SignalSignal Generated: Generated: Non- Endogenous Endogenous Version VersionAssay (Relative (Relative Percent Receptor Mutation Utilized LightUnits) Light Units) Difference hATL F239K SRF-LUC 34 137 75%↑ AT2K2551C3SRF-LUC 34 127 73%↑ hTDAG8 1225K CRE-LUC 2,715 14,440 81%↑ (293 cells)1225K CRE-LUC 65,681 185,636 65%↑ (293T cells) hH9 F236K CRE-LUC 1,8876,096 69%↑ hGGKB V332K CRE-LUC 785 3,223 76%↑

[0224] C. Cell-based Detection Assay (Example-TDAG8)

[0225] 293 cells were plated-out on 150 mm plates at a density of1.3×10⁷ cells per plate, and were transfected using 12 ug of therespective DNA and 60 ul of Lipofectamine Reagent (BRL) per plate. Thetransfected cells were grown in media containing serum for an assayperformed 24 hours post-transfection. For detection assay performed 48hours post-transfecfion (assay comparing serum and serum-free media; seeFIG. 3), the initial media was changed to either serum or serum-freemedia. The serum-free media was comprised solely of Dulbecco's ModifiedEagle's (DME) High Glucose Medium (Irvine Scientific #9024). In additionto the above DME Medium, the media with serum contained the following:10% Fetal Bovine Serum (Hyclone #SH30071.03), 1% of 100 mM SodiumPyruvate (Irvine Scientific #9334), 1% of 20 mM L-Glutamine (IrvineScientific #9317), and 1% of Penicillin-Streptomycin solution (IrvineScientific #9366).

[0226] A 96-well Adenylyl Cyclase Activation Flashplate™ was used (NEN:#SMP004A). First, 50 ul of the standards for the assay were added to theplate, in duplicate, ranging from concentrations of 50 pmol to zero pmolcAMP per well. The standard cAMP (NEN: #SMP004A) was reconstituted inwater, and serial dilutions were made using 1xPBS (Irvine Scientific:#9240). Next, 50 ul of the stimulation buffer (NEN: #SMP004A) was addedto all wells. In the case of using compounds to measure activation orinactivation of cAMP, 10 ul of each compound, diluted in water, wasadded to its respective well, in triplicate. Various finalconcentrations used range from 1 uM up to 1 mM. Adenosine5′-triphosphate, ATP, (ResearchBiochemicals International: #A-141) andAdenosine 5′-diphosphate, ADP, (Sigma: #A2754) were used in the assay.Next, the 293 cells transfected with the respective cDNA (CMV or TDAG8)were harvested 24 (assay detection in serum media) or 48 hourspost-transfection (assay detection comparing serum and serum-freemedia). The media was aspirated and the cells washed once with 1xPBS.Then 5 ml of 1xPBS was added to the cells along with 3 ml of celldissociation buffer (Sigma: #C-1544). The detached cells weretransferred to a centrifuge tube and centrifuged at room temperature forfive minutes. The supernatant was removed and the cell pellet wasresuspended in an appropriate amount of 1xPBS to obtain a finalconcentration of 2×10⁶ cells permilliliter. To the wells containing thecompound, 50 ul of the cells in 1xPBS (1×10⁵ cells/well) were added. Theplate was incubated on a shaker for 15 minutes at room temperature. Thedetection buffer containing the tracer cAMP was prepared. In 11 ml ofdetection buffer (NEN: #SMP004A), 50 ul (equal to 1 uCi) of [¹²⁵I]cAMP(NEN: #SMP004A) was added. Following incubation, 50 ul of this detectionbuffer containing tracer cAMP was added to each well. The plate wasplaced on a shaker and incubated at room temperature for two hours.Finally, the solution from the wells of the plate were aspirated and theflashplate was counted using the Wallac MicroBeta™ scintillationcounter.

[0227] In FIG. 2A, ATP and ADP bind to endogenous TDAG8 resulting in anincrease of cAMP of about 59% and about 55% respectively. FIG. 2Bevidences ATP and ADP binding to endogenous TDAG8 where endogenous TDAG8was transfected and grown in serum and serum-free medium. ATP binding toendogenous TDAG8 grown in serum media evidences an increase in cAMP ofabout 65%, compared to the endogenous TDAG8 with no compounds; inserum-free media there was an increase of about 68%. ADP binding toendogenous TDAG8 in serum evidences about a 61% increase, while inserum-free ADP binding evidences an increase of about 62% increase. ATPand ADP bind to endogenous TDAG8 with an EC50 value of 139.8 uM and120.5 uM, respectively (data not shown).

[0228] Although the results presented in FIG. 2B indicate substantiallythe same results when serum and serum-free media were compared, ourchoice is to use a serum based media, although a serum-free media canalso be utilized.

Example 6

[0229] GPCR Fusion Protein Preparation

[0230] The design of the constitutively activated GPCR-G protein fusionconstruct was accomplished as follows: both the 5′ and 3′ ends of therat G protein Gsa (long form; Itoh, H. et al., 83 PNAS 3776 (1986)) wereengineered to include a HindIII (5′-AAGCTT-3′) sequence thereon.Following confirmation of the correct sequence (including the flankingHindIII sequences), the entire sequence was shuttled into pcDNA3.1(−)(Invitrogen, cat. no. V795-20) by subcloning using the HindIIIrestriction site of that vector. The correct orientation for the Gsasequence was determined after subcloning into pcDNA3.1(−). The modifiedpcDNA3.1 (−) containing the rat Gsa gene at HindIII sequence was thenverified; this vector was now available as a “universal” Gsa proteinvector. The pcDNA3.1(−) vector contains a variety of well-knownrestriction sites upstream of the HindIII site, thus beneficiallyproviding the ability to insert, upstream of the Gs protein, the codingsequence of an endogenous, constitutively active GPCR. This sameapproach can be utilized to create other “universal” G protein vectors,and, of course, other commercially available or proprietary vectorsknown to the artisan can be utilized—the important criteria is that thesequence for the GPCR be upstream and in-frame with that of the Gprotein.

[0231] TDAG8 couples via Gs, while H9 couples via Gz. For the followingexemplary GPCR Fusion Proteins, fusion to Gsα was accomplished.

[0232] A TDAG8(I225K)-Gsa Fusion Protein construct was made as follows:primers were designed as follows:

[0233] 5′-gatcTCTAGAATGAACAGCACATGTATTGAAG-3′ (SEQ.ID.NO.: 125; sense)

[0234] 5′-ctagGGTACCCGCTCAAGGACCTCTAATFCCATAG-3′ (SEQ. ID.NO.: 126;antisense).

[0235] Nucleotides in lower caps are included as spacers in therestriction sites between the G protein and TDAG8. The sense andanti-sense primers included the restriction sites for XbaI and KpnI,respectively.

[0236] PCR was then utilized to secure the respective receptor sequencesfor fusion within the Gsa universal vector disclosed above, using thefollowing protocol for each: 100 ng cDNA for TDAG8 was added to separatetubes containing 2 ul of each primer (sense and anti-sense), 3 uL of 10mM dNTPs, 10 uL of 10XTaqPlus™ Precision buffer, 1 uL of TaqPlus™Precision polymerase (Stratagene: #600211), and 80 uL of water. Reactiontemperatures and cycle times for TDAG8 were as follows: the initialdenaturing step was done it 94° C. for five minutes, and a cycle of 94°C. for 30 seconds; 55° C. for 30 seconds; 72° C. for two minutes. Afinal extension time was done at 72° C. for ten minutes. PCR product forwas run on a 1% agarose gel and then purified (data not shown). Thepurified product was digested with XbaI and KpnI (New England Biolabs)and the desired inserts purified and ligated into the Gs universalvector at the respective restriction site. The positive clones wasisolated following transformation and determined by restriction enzymedigest; expression using 293 cells was accomplished following theprotocol set forth infra. Each positive clone for TDAG8:Gs-FusionProtein was sequenced to verify correctness.

[0237] GPCR Fusion Proteins comprising non-endogenous, constitutivelyactivated TDAG8(I225K) were analyzed as above and verified forconstitutive activation.

[0238] An H9(F236K)-Gsa Fusion Protein construct was made as follows:primers were designed as follows:

[0239] 5′-TTAgatatcGGGGCCCACCCTAGCGGT-3′ (SEQ.ID.NO.: 145; sense)

[0240] 5′-ggtaccCCCACAGCCATITCATCAGGATC-3′ (SEQ.ID.NO.: 146; antisense).

[0241] Nucleotides in lower caps are included as spacers in therestriction sites between the G protein and H9. The sense and anti-senseprimers included the restriction sites for EcoRV and KpnI, respectivelysuch that spacers (attributed to the restriction sites) exists betweenthe G protein and H9.

[0242] PCR was then utilized to secure the respective receptor sequencesfor fusion within the Gsa universal vector disclosed above, using thefollowing protocol for each: 80 ng cDNA for H9 was added to separatetubes containing 100 ng of each primer (sense and anti-sense), and 45 uLof PCR Supermix™ (Gibco-Brl, LifeTech) (50 ul total reaction volume).Reaction temperatures and cycle times for H9 were as follows: theinitial denaturing step was done it 94° C. for one, and a cycle of 94°C. for 30 seconds; 55° C. for 30 seconds; 72° C. for two minutes. Afinal extension time was done at 72° C. for seven minutes. PCR productfor was run on a 1% agarose gel and then purified (data not shown). Thepurified product was cloned into pCRII-TOPO™ System followed byidentification of positive clones. Positive clones were isolated,digested with EcoRV and KpnI (New England Biolabs) and the desiredinserts were isolated, purified and ligated into the Gs universal vectorat the respective restriction site. The positive clones was isolatedfollowing transformation and determined by restriction enzyme digest;expression using 293 cells was accomplished following the protocol setforth infra. Each positive clone for H9(F236K):Gs-Fusion Protein wassequenced to verify correctness. Membranes were frozen (−80° C.) untilutilized.

[0243] To ascertain the ability of measuring a cAMP response mediated bythe Gs protein (even though H9 couples with Gz), the following cAMPmembrane assay was utilized, based upon an NEN Adenyl Cyclase ActivationFlahplate™ Assay kit (96 well format). “Binding Buffer” consisted of 10mM HEPES, 100 mM NaCl and 100 mM MgCl (ph 7.4). “Regeneration Buffer”was prepared in Binding Buffer and consisted of 20 mM phosphocreatine,20 U creatine phosphokinase, 20 uM GTP, 0.2 mM ATP, and 0.6 mM IBMX.“cAMP Standards” were prepared in Binding Buffer as follows: cAMP StockAdded to Final Assay Concentration (5,000 pmol/ml in 2 ml H₂O) indictedamount of Binding (50 ul into 100 ul) in ul Buffer to achieve indicatedpmol/well A 250 1 ml 50 B 500 of A 500 ul 25 C 500 of B 500 ul 12.5 D500 of C 750 ul 5.0 E 500 of D 500 ul 2.5 F 500 of E 500 ul 1.25 G 500of F 750 ul 0.5

[0244] Frozen membranes (both pCMV as control and the non-endogenousH(-Gs Fusion Protein) were thawed (on ice at room temperature until insolution). Membranes were homogenized with a polytron until insuspension (2×15 seconds). Membrane protein concentration was determinedusing the Bradford Assay Protocol (see infra). Membrane concentrationwas diluted to 0.5 mg/ml in Regeneration Buffer (final assayconcentration −25 ug/well). Thereafter, 50 ul of Binding Buffer wasadded to each well. For control, 50 ul/well of cAMP standard was addedto wells 11 and 12 A-G, with Binding Buffer alone to 12H (on the 96-wellformat). Thereafter, 50 ul/well of protein was added to the wells andincubated at room temperature (on shaker) for 60 min. 100 ul[¹²⁵I]cAMPin Detection Buffer (see infra) was added to each well (final −50ul[¹²⁵I]cAMP into 11 ml Detection Buffer). These were incubated for 2hrs at room temperature. Plates were aspirated with an 8 channelmanifold and sealed with plate covers. Results (pmoles cAMP bound) wereread in a Wallac™ 1450 on “prot #15). Results are presented in FIG. 3.

[0245] The results presented in FIG. 3 indicate that the Gs coupledfusion was able to “drive” the cyclase reaction such that measurement ofthe consitutive activation of H9(F236K) was viable. Based upon theseresults, the direct identification of candidate compounds that areinverse agonists, agonists and partial agonists is possible using acyclase-based assay.

Example 6

[0246] Protocol: Direct Identification of Inverse Agonists and AgonistsUsing [³⁵S]GTPγS

[0247] Although we have utilized endogenous, constitutively active GPCRSfor the direct identification of candidate compounds as, e.g., inverseagonists, for reasons that are not altogether understood, intra-assayvariation can become exacerbated. Preferably, then, a GPCR FusionProtein, as disclosed above, is also utilized with a non-endogenous,constitutively activated GPCR. We have determined that when such aprotein is used, intra-assay variation appears to be substantiallystabilized, whereby an effective signal-to-noise ratio is obtained. Thishas the beneficial result of allowing for a more robust identificationof candidate compounds. Thus, it is preferred that for directidentification, a GPCR Fusion Protein be used and that when utilized,the following assay protocols be utilized.

[0248] Membrane Preparation

[0249] Membranes comprising the non-endogenous, constitutively activeorphan GPCR Fusion Protein of interest and for use in the directidentification of candidate compounds as inverse agonists, agonists orpartial agonists are preferably prepared as follows:

[0250] a. Materials

[0251] “Membrane Scrape Buffer” is comprised of 20 mM HEPES and 10 mMEDTA, pH 7.4; “Membrane Wash Buffer” is comprised of 20 mM HEPES and 0.1mM EDTA, pH 7.4; “Binding Buffer” is comprised of 20 mM HEPES, 100 mMNaCl, and 10 mM MgCl₂, pH 7.4

[0252] b. Procedure

[0253] All materials are kept on ice throughout the procedure. Firstly,the media is aspirated from a confluent monolayer of cells, followed byrinse with 10 ml cold PBS, followed by aspiration. Thereafter, 5 ml ofMembrane Scrape Buffer is added to scrape cells; this is followed bytransfer of cellular extract into 50 ml centrifuge tubes (centrifuged at20,000 rpm for 17 minutes at 4° C.). Thereafter, the supernatant isaspirated and the pellet is resuspended in 30 ml Membrane Wash Bufferfollowed by centrifuge at 20,000 rpm for 17 minutes at 4° C. Thesupernatant is then aspirated and the pellet resuspended in BindingBuffer. This is then homogenized using a Brinkman polytron™ homogenizer(15-20 second bursts until the all material is in suspension). This isreferred to herein as “Membrane Protein”.

[0254] Bradford Protein Assay

[0255] Following the homogenization, protein concentration of themembranes is determined using the Bradford Protein Assay (protein can bediluted to about 1.5 mg/ml, aliquoted and frozen (−80° C.) for lateruse; when frozen, protocol for use is as follows: on the day of theassay, frozen Membrane Protein is thawed at room temperature, followedby vortex and then homogenized with a polytron at about 12×1,000 rpm forabout 5-10 seconds; it is noted that for multiple preparations, thehomogenizer should be thoroughly cleaned between homoginezation ofdifferent preparations).

[0256] a. Materials

[0257] Binding Buffer (as per above); Bradford Dye Reagent; BradfordProtein Standard are utilized, following manufacturer instructions(Biorad, cat. no. 500-0006).

[0258] b. Procedure

[0259] Duplicate tubes are prepared, one including the membrane, and oneas a control “blank” . Each contained 800 ul Binding Buffer. Thereafter,10 ul of Bradford Protein Standard (1 mg/ml) is added to each tube, and10 ul of membrane Protein is then added to just one tube (not theblank). Thereafter, 200 ul of Bradford Dye Reagent is added to eachtube, followed by vortex of each. After five (5) minutes, the tubes werere-vortexed and the material therein is transferred to cuvettes. Thecuvettes are then read using a CECIL 3041 spectrophotometer, atwavelength 595.

[0260] Direct Identification Assay

[0261] a. Materials

[0262] GDP Buffer consists of 37.5 ml Binding Buffer and 2 mg GDP(Sigma, cat. no. G-7127), followed by a series of dilutions in BindingBuffer to obtain 0.2 uM GDP (final concentration of GDP in each well was0.1 uM GDP); each well comprising a candidate compound, has a finalvolume of 200 ul consisting of 100 ul GDP Buffer (final concentration,0.1 uM GDP), 50 ul Membrane Protein in Binding Buffer, and 50 ul[³⁵S]GTPγS (0.6 nM) in Binding Buffer (2.5 ul [³⁵S]GTPγS per 10 mlBinding Buffer).

[0263] b. Procedure

[0264] Candidate compounds are preferably screened using a 96-well plateformat (these can be frozen at −80° C.). Membrane Protein (or membraneswith expression vector excluding the GPCR Fusion Protein, as control),are homogenized briefly until in suspension. Protein concentration isthen determined using the Bradford Protein Assay set forth above.Membrane Protein (and control) is then diluted to 0.25 mg/ml in BindingBuffer (final assay concentration, 12.5 ug/well). Thereafter, 100 ul GDPBuffer is added to each well of a Wallac Scintistrip™ (Wallac). A 5 ulpin-tool is then used to transfer 5 ul of a candidate compound into suchwell (i.e., 5 ul in total assay volume of 200 ul is a 1:40 ratio suchthat the final screening concentration of the candidate compound is 10uM). Again, to avoid contamination, after each transfer step the pintool should be rinsed in three reservoirs comprising water (1×), ethanol(1×) and water (2×)—excess liquid should be shaken from the tool aftereach rinse and dried with paper and kimwipes. Thereafter, 50 ul ofMembrane Protein is added to each well (a control well comprisingmembranes without the GPCR Fusion Protein is also utilized), andpre-incubated for 5-10 minutes at room temperature. Thereafter, 50 ulof[³⁵S]GTPγS (0.6 nM) in Binding Buffer is added to each well, followedby incubation on a shaker for 60 minutes at room temperature (again, inthis example, plates were covered with foil). The assay is then stoppedby spinning of the plates at 4000 RPM for 15 minutes at 22° C. Theplates are then aspirated with an 8 channel manifold and sealed withplate covers. The plates are then read on a Wallacc 1450 using setting“Prot. #37” (as per manufacturer instructions).

Example 7 Protocol: Confirmation Assay

[0265] Using an independent assay approach to provide confirmation of adirectly identified candidate compound as set forth above, it ispreferred that a confirmation assay then be utilized. In this case, thepreferred confirmation assay is a cyclase-based assay.

[0266] A modified Flash Plate™ Adenylyl Cyclase kit (New EnglandNuclear; Cat. No. SMP004A) is preferably utilized for confirmation ofcandidate compounds directly identified as inverse agonists and agoniststo non-endogenous, constitutively activated orphan GPCRs in accordancewith the following protocol.

[0267] Transfected cells are harvested approximately three days aftertransfection. Membranes are prepared by homogenization of suspendedcells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl₂.Homogenization is performed on ice using a Brinkman Polytron™ forapproximately 10 seconds. The resulting homogenate is centrifuged at49,000× g for 15 minutes at 4° C. The resulting pellet is thenresuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA,homogenized for 10 seconds, followed by centrifugation at 49,000× g for15 minutes at 4° C. The resulting pellet can be stored at −800° C. untilutilized. On the day of direct identification screening, the membranepellet is slowly thawed at room temperature, resuspended in buffercontaining 20 mM HEPES, pH 7.4 and 10 mM MgCL2, to yield a final proteinconcentration of 0.60 mg/ml (the resuspended membranes are placed on iceuntil use).

[0268] cAMP standards and Detection Buffer (comprising 2 μCi of tracer[¹²⁵I cAMP (100 μl] to 11 ml Detection Buffer) are prepared andmaintained in accordance with the manufacturer's instructions. AssayBuffer is prepared fresh for screening and contained 20 mM HEPES, pH7.4, 10 mM MgCl₂, 20 mM phospocreatine (Sigma), 0.1 units/ml creatinephosphokinase (Sigma), 50 μM GTP (Sigma), and 0.2 mM ATP (Sigma); AssayBuffer can be stored on ice until utilized.

[0269] Candidate compounds identified as per above (if frozen, thawed atroom temperature) are added, preferably, to 96-well plate wells (3μl/well; 12 μM final assay concentration), together with 40 μl MembraneProtein (30 μg/well) and 50 μl of Assay Buffer. This admixture is thenincubated for 30 minutes at room temperature, with gentle shaking.

[0270] Following the incubation, 100 μl of Detection Buffer is added toeach well, followed by incubation for 2-24 hours. Plates are thencounted in a Wallac MicroBeta™ plate reader using “Prot. #31” (as permanufacturer instructions).

[0271] It is intended that each of the patents, applications, andprinted publications mentioned in this patent document be herebyincorporated by reference in their entirety.

[0272] As those skilled in the art will appreciate, numerous changes andmodifications may be made to the preferred embodiments of the inventionwithout departing from the spirit of the invention. It is intended thatall such variations fall within the scope of the invention.

[0273] Although a variety of expression vectors are available to thosein the art, for purposes of utilization for both the endogenous andnon-endogenous human GPCRS, it is most preferred that the vectorutilized be pCMV. This vector was deposited with the American TypeCulture Collection (ATCC) on Oct. 13, 1998 (10801 University Blvd.,Manassas, Va. 20110-2209 USA) under the provisions of the BudapestTreaty for the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure. The DNA was testedby the ATCC and determined to be. The ATCC has assigned the followingdeposit number to pCMV: ATCC #203351.

1 146 1 1260 DNA Homo sapiens 1 atggtcttct cggcagtgtt gactgcgttccataccggga catccaacac aacatttgtc 60 gtgtatgaaa acacctacat gaatattacactccctccac cattccagca tcctgacctc 120 agtccattgc ttagatatag ttttgaaaccatggctccca ctggtttgag ttccttgacc 180 gtgaatagta cagctgtgcc cacaacaccagcagcattta agagcctaaa cttgcctctt 240 cagatcaccc tttctgctat aatgatattcattctgtttg tgtcttttct tgggaacttg 300 gttgtttgcc tcatggttta ccaaaaagctgccatgaggt ctgcaattaa catcctcctt 360 gccagcctag cttttgcaga catgttgcttgcagtgctga acatgccctt tgccctggta 420 actattctta ctacccgatg gatttttgggaaattcttct gtagggtatc tgctatgttt 480 ttctggttat ttgtgataga aggagtagccatcctgctca tcattagcat agataggttc 540 cttattatag tccagaggca ggataagctaaacccatata gagctaaggt tctgattgca 600 gtttcttggg caacttcctt ttgtgtagcttttcctttag ccgtaggaaa ccccgacctg 660 cagatacctt cccgagctcc ccagtgtgtgtttgggtaca caaccaatcc aggctaccag 720 gcttatgtga ttttgatttc tctcatttctttcttcatac ccttcctggt aatactgtac 780 tcatttatgg gcatactcaa cacccttcggcacaatgcct tgaggatcca tagctaccct 840 gaaggtatat gcctcagcca ggccagcaaactgggtctca tgagtctgca gagacctttc 900 cagatgagca ttgacatggg ctttaaaacacgtgccttca ccactatttt gattctcttt 960 gctgtcttca ttgtctgctg ggccccattcaccacttaca gccttgtggc aacattcagt 1020 aagcactttt actatcagca caacttttttgagattagca cctggctact gtggctctgc 1080 tacctcaagt ctgcattgaa tccgctgatctactactgga ggattaagaa attccatgat 1140 gcttgcctgg acatgatgcc taagtccttcaagtttttgc cgcagctccc tggtcacaca 1200 aagcgacgga tacgtcctag tgctgtctatgtgtgtgggg aacatcggac ggtggtgtga 1260 2 419 PRT Homo sapiens 2 Met ValPhe Ser Ala Val Leu Thr Ala Phe His Thr Gly Thr Ser Asn 1 5 10 15 ThrThr Phe Val Val Tyr Glu Asn Thr Tyr Met Asn Ile Thr Leu Pro 20 25 30 ProPro Phe Gln His Pro Asp Leu Ser Pro Leu Leu Arg Tyr Ser Phe 35 40 45 GluThr Met Ala Pro Thr Gly Leu Ser Ser Leu Thr Val Asn Ser Thr 50 55 60 AlaVal Pro Thr Thr Pro Ala Ala Phe Lys Ser Leu Asn Leu Pro Leu 65 70 75 80Gln Ile Thr Leu Ser Ala Ile Met Ile Phe Ile Leu Phe Val Ser Phe 85 90 95Leu Gly Asn Leu Val Val Cys Leu Met Val Tyr Gln Lys Ala Ala Met 100 105110 Arg Ser Ala Ile Asn Ile Leu Leu Ala Ser Leu Ala Phe Ala Asp Met 115120 125 Leu Leu Ala Val Leu Asn Met Pro Phe Ala Leu Val Thr Ile Leu Thr130 135 140 Thr Arg Trp Ile Phe Gly Lys Phe Phe Cys Arg Val Ser Ala MetPhe 145 150 155 160 Phe Trp Leu Phe Val Ile Glu Gly Val Ala Ile Leu LeuIle Ile Ser 165 170 175 Ile Asp Arg Phe Leu Ile Ile Val Gln Arg Gln AspLys Leu Asn Pro 180 185 190 Tyr Arg Ala Lys Val Leu Ile Ala Val Ser TrpAla Thr Ser Phe Cys 195 200 205 Val Ala Phe Pro Leu Ala Val Gly Asn ProAsp Leu Gln Ile Pro Ser 210 215 220 Arg Ala Pro Gln Cys Val Phe Gly TyrThr Thr Asn Pro Gly Tyr Gln 225 230 235 240 Ala Tyr Val Ile Leu Ile SerLeu Ile Ser Phe Phe Ile Pro Phe Leu 245 250 255 Val Ile Leu Tyr Ser PheMet Gly Ile Leu Asn Thr Leu Arg His Asn 260 265 270 Ala Leu Arg Ile HisSer Tyr Pro Glu Gly Ile Cys Leu Ser Gln Ala 275 280 285 Ser Lys Leu GlyLeu Met Ser Leu Gln Arg Pro Phe Gln Met Ser Ile 290 295 300 Asp Met GlyPhe Lys Thr Arg Ala Phe Thr Thr Ile Leu Ile Leu Phe 305 310 315 320 AlaVal Phe Ile Val Cys Trp Ala Pro Phe Thr Thr Tyr Ser Leu Val 325 330 335Ala Thr Phe Ser Lys His Phe Tyr Tyr Gln His Asn Phe Phe Glu Ile 340 345350 Ser Thr Trp Leu Leu Trp Leu Cys Tyr Leu Lys Ser Ala Leu Asn Pro 355360 365 Leu Ile Tyr Tyr Trp Arg Ile Lys Lys Phe His Asp Ala Cys Leu Asp370 375 380 Met Met Pro Lys Ser Phe Lys Phe Leu Pro Gln Leu Pro Gly HisThr 385 390 395 400 Lys Arg Arg Ile Arg Pro Ser Ala Val Tyr Val Cys GlyGlu His Arg 405 410 415 Thr Val Val 3 1119 DNA Homo sapiens 3 atgttagccaacagctcctc aaccaacagt tctgttctcc cgtgtcctga ctaccgacct 60 acccaccgcctgcacttggt ggtctacagc ttggtgctgg ctgccgggct ccccctcaac 120 gcgctagccctctgggtctt cctgcgcgcg ctgcgcgtgc actcggtggt gagcgtgtac 180 atgtgtaacctggcggccag cgacctgctc ttcaccctct cgctgcccgt tcgtctctcc 240 tactacgcactgcaccactg gcccttcccc gacctcctgt gccagacgac gggcgccatc 300 ttccagatgaacatgtacgg cagctgcatc ttcctgatgc tcatcaacgt ggaccgctac 360 gccgccatcgtgcacccgct gcgactgcgc cacctgcggc ggccccgcgt ggcgcggctg 420 ctctgcctgggcgtgtgggc gctcatcctg gtgtttgccg tgcccgccgc ccgcgtgcac 480 aggccctcgcgttgccgcta ccgggacctc gaggtgcgcc tatgcttcga gagcttcagc 540 gacgagctgtggaaaggcag gctgctgccc ctcgtgctgc tggccgaggc gctgggcttc 600 ctgctgcccctggcggcggt ggtctactcg tcgggccgag tcttctggac gctggcgcgc 660 cccgacgccacgcagagcca gcggcggcgg aagaccgtgc gcctcctgct ggctaacctc 720 gtcatcttcctgctgtgctt cgtgccctac aacagcacgc tggcggtcta cgggctgctg 780 cggagcaagctggtggcggc cagcgtgcct gcccgcgatc gcgtgcgcgg ggtgctgatg 840 gtgatggtgctgctggccgg cgccaactgc gtgctggacc cgctggtgta ctactttagc 900 gccgagggcttccgcaacac cctgcgcggc ctgggcactc cgcaccgggc caggacctcg 960 gccaccaacgggacgcgggc ggcgctcgcg caatccgaaa ggtccgccgt caccaccgac 1020 gccaccaggccggatgccgc cagtcagggg ctgctccgac cctccgactc ccactctctg 1080 tcttccttcacacagtgtcc ccaggattcc gccctctga 1119 4 372 PRT Homo sapiens 4 Met LeuAla Asn Ser Ser Ser Thr Asn Ser Ser Val Leu Pro Cys Pro 1 5 10 15 AspTyr Arg Pro Thr His Arg Leu His Leu Val Val Tyr Ser Leu Val 20 25 30 LeuAla Ala Gly Leu Pro Leu Asn Ala Leu Ala Leu Trp Val Phe Leu 35 40 45 ArgAla Leu Arg Val His Ser Val Val Ser Val Tyr Met Cys Asn Leu 50 55 60 AlaAla Ser Asp Leu Leu Phe Thr Leu Ser Leu Pro Val Arg Leu Ser 65 70 75 80Tyr Tyr Ala Leu His His Trp Pro Phe Pro Asp Leu Leu Cys Gln Thr 85 90 95Thr Gly Ala Ile Phe Gln Met Asn Met Tyr Gly Ser Cys Ile Phe Leu 100 105110 Met Leu Ile Asn Val Asp Arg Tyr Ala Ala Ile Val His Pro Leu Arg 115120 125 Leu Arg His Leu Arg Arg Pro Arg Val Ala Arg Leu Leu Cys Leu Gly130 135 140 Val Trp Ala Leu Ile Leu Val Phe Ala Val Pro Ala Ala Arg ValHis 145 150 155 160 Arg Pro Ser Arg Cys Arg Tyr Arg Asp Leu Glu Val ArgLeu Cys Phe 165 170 175 Glu Ser Phe Ser Asp Glu Leu Trp Lys Gly Arg LeuLeu Pro Leu Val 180 185 190 Leu Leu Ala Glu Ala Leu Gly Phe Leu Leu ProLeu Ala Ala Val Val 195 200 205 Tyr Ser Ser Gly Arg Val Phe Trp Thr LeuAla Arg Pro Asp Ala Thr 210 215 220 Gln Ser Gln Arg Arg Arg Lys Thr ValArg Leu Leu Leu Ala Asn Leu 225 230 235 240 Val Ile Phe Leu Leu Cys PheVal Pro Tyr Asn Ser Thr Leu Ala Val 245 250 255 Tyr Gly Leu Leu Arg SerLys Leu Val Ala Ala Ser Val Pro Ala Arg 260 265 270 Asp Arg Val Arg GlyVal Leu Met Val Met Val Leu Leu Ala Gly Ala 275 280 285 Asn Cys Val LeuAsp Pro Leu Val Tyr Tyr Phe Ser Ala Glu Gly Phe 290 295 300 Arg Asn ThrLeu Arg Gly Leu Gly Thr Pro His Arg Ala Arg Thr Ser 305 310 315 320 AlaThr Asn Gly Thr Arg Ala Ala Leu Ala Gln Ser Glu Arg Ser Ala 325 330 335Val Thr Thr Asp Ala Thr Arg Pro Asp Ala Ala Ser Gln Gly Leu Leu 340 345350 Arg Pro Ser Asp Ser His Ser Leu Ser Ser Phe Thr Gln Cys Pro Gln 355360 365 Asp Ser Ala Leu 370 5 1107 DNA Homo sapiens 5 atggccaactccacagggct gaacgcctca gaagtcgcag gctcgttggg gttgatcctg 60 gcagctgtcgtggaggtggg ggcactgctg ggcaacggcg cgctgctggt cgtggtgctg 120 cgcacgccgggactgcgcga cgcgctctac ctggcgcacc tgtgcgtcgt ggacctgctg 180 gcggccgcctccatcatgcc gctgggcctg ctggccgcac cgccgcccgg gctgggccgc 240 gtgcgcctgggccccgcgcc atgccgcgcc gctcgcttcc tctccgccgc tctgctgccg 300 gcctgcacgctcggggtggc cgcacttggc ctggcacgct accgcctcat cgtgcacccg 360 ctgcggccaggctcgcggcc gccgcctgtg ctcgtgctca ccgccgtgtg ggccgcggcg 420 ggactgctgggcgcgctctc cctgctcggc ccgccgcccg caccgccccc tgctcctgct 480 cgctgctcggtcctggctgg gggcctcggg cccttccggc cgctctgggc cctgctggcc 540 ttcgcgctgcccgccctcct gctgctcggc gcctacggcg gcatcttcgt ggtggcgcgt 600 cgcgctgccctgaggccccc acggccggcg cgcgggtccc gactccgctc ggactctctg 660 gatagccgcctttccatctt gccgccgctc cggcctcgcc tgcccggggg caaggcggcc 720 ctggccccagcgctggccgt gggccaattt gcagcctgct ggctgcctta tggctgcgcg 780 tgcctggcgcccgcagcgcg ggccgcggaa gccgaagcgg ctgtcacctg ggtcgcctac 840 tcggccttcgcggctcaccc cttcctgtac gggctgctgc agcgccccgt gcgcttggca 900 ctgggccgcctctctcgccg tgcactgcct ggacctgtgc gggcctgcac tccgcaagcc 960 tggcacccgcgggcactctt gcaatgcctc cagagacccc cagagggccc tgccgtaggc 1020 ccttctgaggctccagaaca gacccccgag ttggcaggag ggcggagccc cgcataccag 1080 gggccacctgagagttctct ctcctga 1107 6 368 PRT Homo sapiens 6 Met Ala Asn Ser Thr GlyLeu Asn Ala Ser Glu Val Ala Gly Ser Leu 1 5 10 15 Gly Leu Ile Leu AlaAla Val Val Glu Val Gly Ala Leu Leu Gly Asn 20 25 30 Gly Ala Leu Leu ValVal Val Leu Arg Thr Pro Gly Leu Arg Asp Ala 35 40 45 Leu Tyr Leu Ala HisLeu Cys Val Val Asp Leu Leu Ala Ala Ala Ser 50 55 60 Ile Met Pro Leu GlyLeu Leu Ala Ala Pro Pro Pro Gly Leu Gly Arg 65 70 75 80 Val Arg Leu GlyPro Ala Pro Cys Arg Ala Ala Arg Phe Leu Ser Ala 85 90 95 Ala Leu Leu ProAla Cys Thr Leu Gly Val Ala Ala Leu Gly Leu Ala 100 105 110 Arg Tyr ArgLeu Ile Val His Pro Leu Arg Pro Gly Ser Arg Pro Pro 115 120 125 Pro ValLeu Val Leu Thr Ala Val Trp Ala Ala Ala Gly Leu Leu Gly 130 135 140 AlaLeu Ser Leu Leu Gly Pro Pro Pro Ala Pro Pro Pro Ala Pro Ala 145 150 155160 Arg Cys Ser Val Leu Ala Gly Gly Leu Gly Pro Phe Arg Pro Leu Trp 165170 175 Ala Leu Leu Ala Phe Ala Leu Pro Ala Leu Leu Leu Leu Gly Ala Tyr180 185 190 Gly Gly Ile Phe Val Val Ala Arg Arg Ala Ala Leu Arg Pro ProArg 195 200 205 Pro Ala Arg Gly Ser Arg Leu Arg Ser Asp Ser Leu Asp SerArg Leu 210 215 220 Ser Ile Leu Pro Pro Leu Arg Pro Arg Leu Pro Gly GlyLys Ala Ala 225 230 235 240 Leu Ala Pro Ala Leu Ala Val Gly Gln Phe AlaAla Cys Trp Leu Pro 245 250 255 Tyr Gly Cys Ala Cys Leu Ala Pro Ala AlaArg Ala Ala Glu Ala Glu 260 265 270 Ala Ala Val Thr Trp Val Ala Tyr SerAla Phe Ala Ala His Pro Phe 275 280 285 Leu Tyr Gly Leu Leu Gln Arg ProVal Arg Leu Ala Leu Gly Arg Leu 290 295 300 Ser Arg Arg Ala Leu Pro GlyPro Val Arg Ala Cys Thr Pro Gln Ala 305 310 315 320 Trp His Pro Arg AlaLeu Leu Gln Cys Leu Gln Arg Pro Pro Glu Gly 325 330 335 Pro Ala Val GlyPro Ser Glu Ala Pro Glu Gln Thr Pro Glu Leu Ala 340 345 350 Gly Gly ArgSer Pro Ala Tyr Gln Gly Pro Pro Glu Ser Ser Leu Ser 355 360 365 7 1008DNA Homo sapiens 7 atggaatcat ctttctcatt tggagtgatc cttgctgtcctggcctccct catcattgct 60 actaacacac tagtggctgt ggctgtgctg ctgttgatccacaagaatga tggtgtcagt 120 ctctgcttca ccttgaatct ggctgtggct gacaccttgattggtgtggc catctctggc 180 ctactcacag accagctctc cagcccttct cggcccacacagaagaccct gtgcagcctg 240 cggatggcat ttgtcacttc ctccgcagct gcctctgtcctcacggtcat gctgatcacc 300 tttgacaggt accttgccat caagcagccc ttccgctacttgaagatcat gagtgggttc 360 gtggccgggg cctgcattgc cgggctgtgg ttagtgtcttacctcattgg cttcctccca 420 ctcggaatcc ccatgttcca gcagactgcc tacaaagggcagtgcagctt ctttgctgta 480 tttcaccctc acttcgtgct gaccctctcc tgcgttggcttcttcccagc catgctcctc 540 tttgtcttct tctactgcga catgctcaag attgcctccatgcacagcca gcagattcga 600 aagatggaac atgcaggagc catggctgga ggttatcgatccccacggac tcccagcgac 660 ttcaaagctc tccgtactgt gtctgttctc attgggagctttgctctatc ctggaccccc 720 ttccttatca ctggcattgt gcaggtggcc tgccaggagtgtcacctcta cctagtgctg 780 gaacggtacc tgtggctgct cggcgtgggc aactccctgctcaacccact catctatgcc 840 tattggcaga aggaggtgcg actgcagctc taccacatggccctaggagt gaagaaggtg 900 ctcacctcat tcctcctctt tctctcggcc aggaattgtggcccagagag gcccagggaa 960 agttcctgtc acatcgtcac tatctccagc tcagagtttgatggctaa 1008 8 335 PRT Homo sapiens 8 Met Glu Ser Ser Phe Ser Phe GlyVal Ile Leu Ala Val Leu Ala Ser 1 5 10 15 Leu Ile Ile Ala Thr Asn ThrLeu Val Ala Val Ala Val Leu Leu Leu 20 25 30 Ile His Lys Asn Asp Gly ValSer Leu Cys Phe Thr Leu Asn Leu Ala 35 40 45 Val Ala Asp Thr Leu Ile GlyVal Ala Ile Ser Gly Leu Leu Thr Asp 50 55 60 Gln Leu Ser Ser Pro Ser ArgPro Thr Gln Lys Thr Leu Cys Ser Leu 65 70 75 80 Arg Met Ala Phe Val ThrSer Ser Ala Ala Ala Ser Val Leu Thr Val 85 90 95 Met Leu Ile Thr Phe AspArg Tyr Leu Ala Ile Lys Gln Pro Phe Arg 100 105 110 Tyr Leu Lys Ile MetSer Gly Phe Val Ala Gly Ala Cys Ile Ala Gly 115 120 125 Leu Trp Leu ValSer Tyr Leu Ile Gly Phe Leu Pro Leu Gly Ile Pro 130 135 140 Met Phe GlnGln Thr Ala Tyr Lys Gly Gln Cys Ser Phe Phe Ala Val 145 150 155 160 PheHis Pro His Phe Val Leu Thr Leu Ser Cys Val Gly Phe Phe Pro 165 170 175Ala Met Leu Leu Phe Val Phe Phe Tyr Cys Asp Met Leu Lys Ile Ala 180 185190 Ser Met His Ser Gln Gln Ile Arg Lys Met Glu His Ala Gly Ala Met 195200 205 Ala Gly Gly Tyr Arg Ser Pro Arg Thr Pro Ser Asp Phe Lys Ala Leu210 215 220 Arg Thr Val Ser Val Leu Ile Gly Ser Phe Ala Leu Ser Trp ThrPro 225 230 235 240 Phe Leu Ile Thr Gly Ile Val Gln Val Ala Cys Gln GluCys His Leu 245 250 255 Tyr Leu Val Leu Glu Arg Tyr Leu Trp Leu Leu GlyVal Gly Asn Ser 260 265 270 Leu Leu Asn Pro Leu Ile Tyr Ala Tyr Trp GlnLys Glu Val Arg Leu 275 280 285 Gln Leu Tyr His Met Ala Leu Gly Val LysLys Val Leu Thr Ser Phe 290 295 300 Leu Leu Phe Leu Ser Ala Arg Asn CysGly Pro Glu Arg Pro Arg Glu 305 310 315 320 Ser Ser Cys His Ile Val ThrIle Ser Ser Ser Glu Phe Asp Gly 325 330 335 9 1413 DNA Homo sapiens 9atggacacta ccatggaagc tgacctgggt gccactggcc acaggccccg cacagagctt 60gatgatgagg actcctaccc ccaaggtggc tgggacacgg tcttcctggt ggccctgctg 120ctccttgggc tgccagccaa tgggttgatg gcgtggctgg ccggctccca ggcccggcat 180ggagctggca cgcgtctggc gctgctcctg ctcagcctgg ccctctctga cttcttgttc 240ctggcagcag cggccttcca gatcctagag atccggcatg ggggacactg gccgctgggg 300acagctgcct gccgcttcta ctacttccta tggggcgtgt cctactcctc cggcctcttc 360ctgctggccg ccctcagcct cgaccgctgc ctgctggcgc tgtgcccaca ctggtaccct 420gggcaccgcc cagtccgcct gcccctctgg gtctgcgccg gtgtctgggt gctggccaca 480ctcttcagcg tgccctggct ggtcttcccc gaggctgccg tctggtggta cgacctggtc 540atctgcctgg acttctggga cagcgaggag ctgtcgctga ggatgctgga ggtcctgggg 600ggcttcctgc ctttcctcct gctgctcgtc tgccacgtgc tcacccaggc cacagcctgt 660cgcacctgcc accgccaaca gcagcccgca gcctgccggg gcttcgcccg tgtggccagg 720accattctgt cagcctatgt ggtcctgagg ctgccctacc agctggccca gctgctctac 780ctggccttcc tgtgggacgt ctactctggc tacctgctct gggaggccct ggtctactcc 840gactacctga tcctactcaa cagctgcctc agccccttcc tctgcctcat ggccagtgcc 900gacctccgga ccctgctgcg ctccgtgctc tcgtccttcg cggcagctct ctgcgaggag 960cggccgggca gcttcacgcc cactgagcca cagacccagc tagattctga gggtccaact 1020ctgccagagc cgatggcaga ggcccagtca cagatggatc ctgtggccca gcctcaggtg 1080aaccccacac tccagccacg atcggatccc acagctcagc cacagctgaa ccctacggcc 1140cagccacagt cggatcccac agcccagcca cagctgaacc tcatggccca gccacagtca 1200gattctgtgg cccagccaca ggcagacact aacgtccaga cccctgcacc tgctgccagt 1260tctgtgccca gtccctgtga tgaagcttcc ccaaccccat cctcgcatcc taccccaggg 1320gcccttgagg acccagccac acctcctgcc tctgaaggag aaagccccag cagcaccccg 1380ccagaggcgg ccccgggcgc aggccccacg tga 1413 10 468 PRT Homo sapiens 10 MetAsp Thr Thr Met Glu Ala Asp Leu Gly Ala Thr Gly His Arg Pro 1 5 10 15Arg Thr Glu Leu Asp Asp Glu Asp Ser Tyr Pro Gln Gly Gly Trp Asp 20 25 30Thr Val Phe Leu Val Ala Leu Leu Leu Leu Gly Leu Pro Ala Asn Gly 35 40 45Leu Met Ala Trp Leu Ala Gly Ser Gln Ala Arg His Gly Ala Gly Thr 50 55 60Arg Leu Ala Leu Leu Leu Leu Ser Leu Ala Leu Ser Asp Phe Leu Phe 65 70 7580 Leu Ala Ala Ala Ala Phe Gln Ile Leu Glu Ile Arg His Gly Gly His 85 9095 Trp Pro Leu Gly Thr Ala Ala Cys Arg Phe Tyr Tyr Phe Leu Trp Gly 100105 110 Val Ser Tyr Ser Ser Gly Leu Phe Leu Leu Ala Ala Leu Ser Leu Asp115 120 125 Arg Cys Leu Leu Ala Leu Cys Pro His Trp Tyr Pro Gly His ArgPro 130 135 140 Val Arg Leu Pro Leu Trp Val Cys Ala Gly Val Trp Val LeuAla Thr 145 150 155 160 Leu Phe Ser Val Pro Trp Leu Val Phe Pro Glu AlaAla Val Trp Trp 165 170 175 Tyr Asp Leu Val Ile Cys Leu Asp Phe Trp AspSer Glu Glu Leu Ser 180 185 190 Leu Arg Met Leu Glu Val Leu Gly Gly PheLeu Pro Phe Leu Leu Leu 195 200 205 Leu Val Cys His Val Leu Thr Gln AlaThr Arg Thr Cys His Arg Gln 210 215 220 Gln Gln Pro Ala Ala Cys Arg GlyPhe Ala Arg Val Ala Arg Thr Ile 225 230 235 240 Leu Ser Ala Tyr Val ValLeu Arg Leu Pro Tyr Gln Leu Ala Gln Leu 245 250 255 Leu Tyr Leu Ala PheLeu Trp Asp Val Tyr Ser Gly Tyr Leu Leu Trp 260 265 270 Glu Ala Leu ValTyr Ser Asp Tyr Leu Ile Leu Leu Asn Ser Cys Leu 275 280 285 Ser Pro PheLeu Cys Leu Met Ala Ser Ala Asp Leu Arg Thr Leu Leu 290 295 300 Arg SerVal Leu Ser Ser Phe Ala Ala Ala Leu Cys Glu Glu Arg Pro 305 310 315 320Gly Ser Phe Thr Pro Thr Glu Pro Gln Thr Gln Leu Asp Ser Glu Gly 325 330335 Pro Thr Leu Pro Glu Pro Met Ala Glu Ala Gln Ser Gln Met Asp Pro 340345 350 Val Ala Gln Pro Gln Val Asn Pro Thr Leu Gln Pro Arg Ser Asp Pro355 360 365 Thr Ala Gln Pro Gln Leu Asn Pro Thr Ala Gln Pro Gln Ser AspPro 370 375 380 Thr Ala Gln Pro Gln Leu Asn Leu Met Ala Gln Pro Gln SerAsp Ser 385 390 395 400 Val Ala Gln Pro Gln Ala Asp Thr Asn Val Gln ThrPro Ala Pro Ala 405 410 415 Ala Ser Ser Val Pro Ser Pro Cys Asp Glu AlaSer Pro Thr Pro Ser 420 425 430 Ser His Pro Thr Pro Gly Ala Leu Glu AspPro Ala Thr Pro Pro Ala 435 440 445 Ser Glu Gly Glu Ser Pro Ser Ser ThrPro Pro Glu Ala Ala Pro Gly 450 455 460 Ala Gly Pro Thr 465 11 1248 DNAHomo sapiens 11 atgtcaggga tggaaaaact tcagaatgct tcctggatct accagcagaaactagaagat 60 ccattccaga aacacctgaa cagcaccgag gagtatctgg ccttcctctgcggacctcgg 120 cgcagccact tcttcctccc cgtgtctgtg gtgtatgtgc caatttttgtggtgggggtc 180 attggcaatg tcctggtgtg cctggtgatt ctgcagcacc aggctatgaagacgcccacc 240 aactactacc tcttcagcct ggcggtctct gacctcctgg tcctgctccttggaatgccc 300 ctggaggtct atgagatgtg gcgcaactac cctttcttgt tcgggcccgtgggctgctac 360 ttcaagacgg ccctctttga gaccgtgtgc ttcgcctcca tcctcagcatcaccaccgtc 420 agcgtggagc gctacgtggc catcctacac ccgttccgcg ccaaactgcagagcacccgg 480 cgccgggccc tcaggatcct cggcatcgtc tggggcttct ccgtgctcttctccctgccc 540 aacaccagca tccatggcat caagttccac tacttcccca atgggtccctggtcccaggt 600 tcggccacct gtacggtcat caagcccatg tggatctaca atttcatcatccaggtcacc 660 tccttcctat tctacctcct ccccatgact gtcatcagtg tcctctactacctcatggca 720 ctcagactaa agaaagacaa atctcttgag gcagatgaag ggaatgcaaatattcaaaga 780 ccctgcagaa aatcagtcaa caagatgctg tttgtcttgg tcttagtgtttgctatctgt 840 tgggccccgt tccacattga ccgactcttc ttcagctttg tggaggagtggagtgaatcc 900 ctggctgctg tgttcaacct cgtccatgtg gtgtcaggtg tcttcttctacctgagctca 960 gctgtcaacc ccattatcta taacctactg tctcgccgct tccaggcagcattccagaat 1020 gtgatctctt ctttccacaa acagtggcac tcccagcatg acccacagttgccacctgcc 1080 cagcggaaca tcttcctgac agaatgccac tttgtggagc tgaccgaagatataggtccc 1140 caattcccat gtcagtcatc catgcacaac tctcacctcc caacagccctctctagtgaa 1200 cagatgtcaa gaacaaacta tcaaagcttc cactttaaca aaacctga1248 12 415 PRT Homo sapiens 12 Met Ser Gly Met Glu Lys Leu Gln Asn AlaSer Trp Ile Tyr Gln Gln 1 5 10 15 Lys Leu Glu Asp Pro Phe Gln Lys HisLeu Asn Ser Thr Glu Glu Tyr 20 25 30 Leu Ala Phe Leu Cys Gly Pro Arg ArgSer His Phe Phe Leu Pro Val 35 40 45 Ser Val Val Tyr Val Pro Ile Phe ValVal Gly Val Ile Gly Asn Val 50 55 60 Leu Val Cys Leu Val Ile Leu Gln HisGln Ala Met Lys Thr Pro Thr 65 70 75 80 Asn Tyr Tyr Leu Phe Ser Leu AlaVal Ser Asp Leu Leu Val Leu Leu 85 90 95 Leu Gly Met Pro Leu Glu Val TyrGlu Met Trp Arg Asn Tyr Pro Phe 100 105 110 Leu Phe Gly Pro Val Gly CysTyr Phe Lys Thr Ala Leu Phe Glu Thr 115 120 125 Val Cys Phe Ala Ser IleLeu Ser Ile Thr Thr Val Ser Val Glu Arg 130 135 140 Tyr Val Ala Ile LeuHis Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg 145 150 155 160 Arg Arg AlaLeu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu 165 170 175 Phe SerLeu Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe 180 185 190 ProAsn Gly Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys 195 200 205Pro Met Trp Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe 210 215220 Tyr Leu Leu Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala 225230 235 240 Leu Arg Leu Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly AsnAla 245 250 255 Asn Ile Gln Arg Pro Cys Arg Lys Ser Val Asn Lys Met LeuPhe Val 260 265 270 Leu Val Leu Val Phe Ala Ile Cys Trp Ala Pro Phe HisIle Asp Arg 275 280 285 Leu Phe Phe Ser Phe Val Glu Glu Trp Ser Glu SerLeu Ala Ala Val 290 295 300 Phe Asn Leu Val His Val Val Ser Gly Val PhePhe Tyr Leu Ser Ser 305 310 315 320 Ala Val Asn Pro Ile Ile Tyr Asn LeuLeu Ser Arg Arg Phe Gln Ala 325 330 335 Ala Phe Gln Asn Val Ile Ser SerPhe His Lys Gln Trp His Ser Gln 340 345 350 His Asp Pro Gln Leu Pro ProAla Gln Arg Asn Ile Phe Leu Thr Glu 355 360 365 Cys His Phe Val Glu LeuThr Glu Asp Ile Gly Pro Gln Phe Pro Cys 370 375 380 Gln Ser Ser Met HisAsn Ser His Leu Pro Thr Ala Leu Ser Ser Glu 385 390 395 400 Gln Met SerArg Thr Asn Tyr Gln Ser Phe His Phe Asn Lys Thr 405 410 415 13 1173 DNAHomo sapiens 13 atgccagata ctaatagcac aatcaattta tcactaagca ctcgtgttactttagcattt 60 tttatgtcct tagtagcttt tgctataatg ctaggaaatg ctttggtcattttagctttt 120 gtggtggaca aaaaccttag acatcgaagt agttattttt ttcttaacttggccatctct 180 gacttctttg tgggtgtgat ctccattcct ttgtacatcc ctcacacgctgttcgaatgg 240 gattttggaa aggaaatctg tgtattttgg ctcactactg actatctgttatgtacagca 300 tctgtatata acattgtcct catcagctat gatcgatacc tgtcagtctcaaatgctgtg 360 tcttatagaa ctcaacatac tggggtcttg aagattgtta ctctgatggtggccgtttgg 420 gtgctggcct tcttagtgaa tgggccaatg attctagttt cagagtcttggaaggatgaa 480 ggtagtgaat gtgaacctgg atttttttcg gaatggtaca tccttgccatcacatcattc 540 ttggaattcg tgatcccagt catcttagtc gcttatttca acatgaatatttattggagc 600 ctgtggaagc gtgatcatct cagtaggtgc caaagccatc ctggactgactgctgtctct 660 tccaacatct gtggacactc attcagaggt agactatctt caaggagatctctttctgca 720 tcgacagaag ttcctgcatc ctttcattca gagagacaga ggagaaagagtagtctcatg 780 ttttcctcaa gaaccaagat gaatagcaat acaattgctt ccaaaatgggttccttctcc 840 caatcagatt ctgtagctct tcaccaaagg gaacatgttg aactgcttagagccaggaga 900 ttagccaagt cactggccat tctcttaggg gtttttgctg tttgctgggctccatattct 960 ctgttcacaa ttgtcctttc attttattcc tcagcaacag gtcctaaatcagtttggtat 1020 agaattgcat tttggcttca gtggttcaat tcctttgtca atcctcttttgtatccattg 1080 tgtcacaagc gctttcaaaa ggctttcttg aaaatatttt gtataaaaaagcaacctcta 1140 ccatcacaac acagtcggtc agtatcttct taa 1173 14 390 PRTHomo sapiens 14 Met Pro Asp Thr Asn Ser Thr Ile Asn Leu Ser Leu Ser ThrArg Val 1 5 10 15 Thr Leu Ala Phe Phe Met Ser Leu Val Ala Phe Ala IleMet Leu Gly 20 25 30 Asn Ala Leu Val Ile Leu Ala Phe Val Val Asp Lys AsnLeu Arg His 35 40 45 Arg Ser Ser Tyr Phe Phe Leu Asn Leu Ala Ile Ser AspPhe Phe Val 50 55 60 Gly Val Ile Ser Ile Pro Leu Tyr Ile Pro His Thr LeuPhe Glu Trp 65 70 75 80 Asp Phe Gly Lys Glu Ile Cys Val Phe Trp Leu ThrThr Asp Tyr Leu 85 90 95 Leu Cys Thr Ala Ser Val Tyr Asn Ile Val Leu IleSer Tyr Asp Arg 100 105 110 Tyr Leu Ser Val Ser Asn Ala Val Ser Tyr ArgThr Gln His Thr Gly 115 120 125 Val Leu Lys Ile Val Thr Leu Met Val AlaVal Trp Val Leu Ala Phe 130 135 140 Leu Val Asn Gly Pro Met Ile Leu ValSer Glu Ser Trp Lys Asp Glu 145 150 155 160 Gly Ser Glu Cys Glu Pro GlyPhe Phe Ser Glu Trp Tyr Ile Leu Ala 165 170 175 Ile Thr Ser Phe Leu GluPhe Val Ile Pro Val Ile Leu Val Ala Tyr 180 185 190 Phe Asn Met Asn IleTyr Trp Ser Leu Trp Lys Arg Asp His Leu Ser 195 200 205 Arg Cys Gln SerHis Pro Gly Leu Thr Ala Val Ser Ser Asn Ile Cys 210 215 220 Gly His SerPhe Arg Gly Arg Leu Ser Ser Arg Arg Ser Leu Ser Ala 225 230 235 240 SerThr Glu Val Pro Ala Ser Phe His Ser Glu Arg Gln Arg Arg Lys 245 250 255Ser Ser Leu Met Phe Ser Ser Arg Thr Lys Met Asn Ser Asn Thr Ile 260 265270 Ala Ser Lys Met Gly Ser Phe Ser Gln Ser Asp Ser Val Ala Leu His 275280 285 Gln Arg Glu His Val Glu Leu Leu Arg Ala Arg Arg Leu Ala Lys Ser290 295 300 Leu Ala Ile Leu Leu Gly Val Phe Ala Val Cys Trp Ala Pro TyrSer 305 310 315 320 Leu Phe Thr Ile Val Leu Ser Phe Tyr Ser Ser Ala ThrGly Pro Lys 325 330 335 Ser Val Trp Tyr Arg Ile Ala Phe Trp Leu Gln TrpPhe Asn Ser Phe 340 345 350 Val Asn Pro Leu Leu Tyr Pro Leu Cys His LysArg Phe Gln Lys Ala 355 360 365 Phe Leu Lys Ile Phe Cys Ile Lys Lys GlnPro Leu Pro Ser Gln His 370 375 380 Ser Arg Ser Val Ser Ser 385 390 1530 DNA Artificial Sequence Novel Sequence 15 ggaaagctta acgatccccaggagcaacat 30 16 31 DNA Artificial Sequence Novel Sequence 16 ctgggatcctacgagagcat ttttcacaca g 31 17 1128 DNA Homo sapiens 17 atggcgaacgcgagcgagcc gggtggcagc ggcggcggcg aggcggccgc cctgggcctc 60 aagctggccacgctcagcct gctgctgtgc gtgagcctag cgggcaacgt gctgttcgcg 120 ctgctgatcgtgcgggagcg cagcctgcac cgcgccccgt actacctgct gctcgacctg 180 tgcctggccgacgggctgcg cgcgctcgcc tgcctcccgg ccgtcatgct ggcggcgcgg 240 cgtgcggcggccgcggcggg ggcgccgccg ggcgcgctgg gctgcaagct gctcgccttc 300 ctggccgcgctcttctgctt ccacgccgcc ttcctgctgc tgggcgtggg cgtcacccgc 360 tacctggccatcgcgcacca ccgcttctat gcagagcgcc tggccggctg gccgtgcgcc 420 gccatgctggtgtgcgccgc ctgggcgctg gcgctggccg cggccttccc gccagtgctg 480 gacggcggtggcgacgacga ggacgcgccg tgcgccctgg agcagcggcc cgacggcgcc 540 cccggcgcgctgggcttcct gctgctgctg gccgtggtgg tgggcgccac gcacctcgtc 600 tacctccgcctgctcttctt catccacgac cgccgcaaga tgcggcccgc gcgcctggtg 660 cccgccgtcagccacgactg gaccttccac ggcccgggcg ccaccggcca ggcggccgcc 720 aactggacggcgggcttcgg ccgcgggccc acgccgcccg cgcttgtggg catccggccc 780 gcagggccgggccgcggcgc gcgccgcctc ctcgtgctgg aagaattcaa gacggagaag 840 aggctgtgcaagatgttcta cgccgtcacg ctgctcttcc tgctcctctg ggggccctac 900 gtcgtggccagctacctgcg ggtcctggtg cggcccggcg ccgtccccca ggcctacctg 960 acggcctccgtgtggctgac cttcgcgcag gccggcatca accccgtcgt gtgcttcctc 1020 ttcaacagggagctgaggga ctgcttcagg gcccagttcc cctgctgcca gagcccccgg 1080 accacccaggcgacccatcc ctgcgacctg aaaggcattg gtttatga 1128 18 375 PRT Homo sapiens18 Met Ala Asn Ala Ser Glu Pro Gly Gly Ser Gly Gly Gly Glu Ala Ala 1 510 15 Ala Leu Gly Leu Lys Leu Ala Thr Leu Ser Leu Leu Leu Cys Val Ser 2025 30 Leu Ala Gly Asn Val Leu Phe Ala Leu Leu Ile Val Arg Glu Arg Ser 3540 45 Leu His Arg Ala Pro Tyr Tyr Leu Leu Leu Asp Leu Cys Leu Ala Asp 5055 60 Gly Leu Arg Ala Leu Ala Cys Leu Pro Ala Val Met Leu Ala Ala Arg 6570 75 80 Arg Ala Ala Ala Ala Ala Gly Ala Pro Pro Gly Ala Leu Gly Cys Lys85 90 95 Leu Leu Ala Phe Leu Ala Ala Leu Phe Cys Phe His Ala Ala Phe Leu100 105 110 Leu Leu Gly Val Gly Val Thr Arg Tyr Leu Ala Ile Ala His HisArg 115 120 125 Phe Tyr Ala Glu Arg Leu Ala Gly Trp Pro Cys Ala Ala MetLeu Val 130 135 140 Cys Ala Ala Trp Ala Leu Ala Leu Ala Ala Ala Phe ProPro Val Leu 145 150 155 160 Asp Gly Gly Gly Asp Asp Glu Asp Ala Pro CysAla Leu Glu Gln Arg 165 170 175 Pro Asp Gly Ala Pro Gly Ala Leu Gly PheLeu Leu Leu Leu Ala Val 180 185 190 Val Val Gly Ala Thr His Leu Val TyrLeu Arg Leu Leu Phe Phe Ile 195 200 205 His Asp Arg Arg Lys Met Arg ProAla Arg Leu Val Pro Ala Val Ser 210 215 220 His Asp Trp Thr Phe His GlyPro Gly Ala Thr Gly Gln Ala Ala Ala 225 230 235 240 Asn Trp Thr Ala GlyPhe Gly Arg Gly Pro Thr Pro Pro Ala Leu Val 245 250 255 Gly Ile Arg ProAla Gly Pro Gly Arg Gly Ala Arg Arg Leu Leu Val 260 265 270 Leu Glu GluPhe Lys Thr Glu Lys Arg Leu Cys Lys Met Phe Tyr Ala 275 280 285 Val ThrLeu Leu Phe Leu Leu Leu Trp Gly Pro Tyr Val Val Ala Ser 290 295 300 TyrLeu Arg Val Leu Val Arg Pro Gly Ala Val Pro Gln Ala Tyr Leu 305 310 315320 Thr Ala Ser Val Trp Leu Thr Phe Ala Gln Ala Gly Ile Asn Pro Val 325330 335 Val Cys Phe Leu Phe Asn Arg Glu Leu Arg Asp Cys Phe Arg Ala Gln340 345 350 Phe Pro Cys Cys Gln Ser Pro Arg Thr Thr Gln Ala Thr His ProCys 355 360 365 Asp Leu Lys Gly Ile Gly Leu 370 375 19 1002 DNA Homosapiens 19 atgaacacca cagtgatgca aggcttcaac agatctgagc ggtgccccagagacactcgg 60 atagtacagc tggtattccc agccctctac acagtggttt tcttgaccggcatcctgctg 120 aatactttgg ctctgtgggt gtttgttcac atccccagct cctccaccttcatcatctac 180 ctcaaaaaca ctttggtggc cgacttgata atgacactca tgcttcctttcaaaatcctc 240 tctgactcac acctggcacc ctggcagctc agagcttttg tgtgtcgtttttcttcggtg 300 atattttatg agaccatgta tgtgggcatc gtgctgttag ggctcatagcctttgacaga 360 ttcctcaaga tcatcagacc tttgagaaat atttttctaa aaaaacctgtttttgcaaaa 420 acggtctcaa tcttcatctg gttctttttg ttcttcatct ccctgccaaatacgatcttg 480 agcaacaagg aagcaacacc atcgtctgtg aaaaagtgtg cttccttaaaggggcctctg 540 gggctgaaat ggcatcaaat ggtaaataac atatgccagt ttattttctggactgttttt 600 atcctaatgc ttgtgtttta tgtggttatt gcaaaaaaag tatatgattcttatagaaag 660 tccaaaagta aggacagaaa aaacaacaaa aagctggaag gcaaagtatttgttgtcgtg 720 gctgtcttct ttgtgtgttt tgctccattt cattttgcca gagttccatatactcacagt 780 caaaccaaca ataagactga ctgtagactg caaaatcaac tgtttattgctaaagaaaca 840 actctctttt tggcagcaac taacatttgt atggatccct taatatacatattcttatgt 900 aaaaaattca cagaaaagct accatgtatg caagggagaa agaccacagcatcaagccaa 960 gaaaatcata gcagtcagac agacaacata accttaggct ga 1002 20333 PRT Homo sapiens 20 Met Asn Thr Thr Val Met Gln Gly Phe Asn Arg SerGlu Arg Cys Pro 1 5 10 15 Arg Asp Thr Arg Ile Val Gln Leu Val Phe ProAla Leu Tyr Thr Val 20 25 30 Val Phe Leu Thr Gly Ile Leu Leu Asn Thr LeuAla Leu Trp Val Phe 35 40 45 Val His Ile Pro Ser Ser Ser Thr Phe Ile IleTyr Leu Lys Asn Thr 50 55 60 Leu Val Ala Asp Leu Ile Met Thr Leu Met LeuPro Phe Lys Ile Leu 65 70 75 80 Ser Asp Ser His Leu Ala Pro Trp Gln LeuArg Ala Phe Val Cys Arg 85 90 95 Phe Ser Ser Val Ile Phe Tyr Glu Thr MetTyr Val Gly Ile Val Leu 100 105 110 Leu Gly Leu Ile Ala Phe Asp Arg PheLeu Lys Ile Ile Arg Pro Leu 115 120 125 Arg Asn Ile Phe Leu Lys Lys ProVal Phe Ala Lys Thr Val Ser Ile 130 135 140 Phe Ile Trp Phe Phe Leu PhePhe Ile Ser Leu Pro Asn Thr Ile Leu 145 150 155 160 Ser Asn Lys Glu AlaThr Pro Ser Ser Val Lys Lys Cys Ala Ser Leu 165 170 175 Lys Gly Pro LeuGly Leu Lys Trp His Gln Met Val Asn Asn Ile Cys 180 185 190 Gln Phe IlePhe Trp Thr Val Phe Ile Leu Met Leu Val Phe Tyr Val 195 200 205 Val IleAla Lys Lys Val Tyr Asp Ser Tyr Arg Lys Ser Lys Ser Lys 210 215 220 AspArg Lys Asn Asn Lys Lys Leu Glu Gly Lys Val Phe Val Val Val 225 230 235240 Ala Val Phe Phe Val Cys Phe Ala Pro Phe His Phe Ala Arg Val Pro 245250 255 Tyr Thr His Ser Gln Thr Asn Asn Lys Thr Asp Cys Arg Leu Gln Asn260 265 270 Gln Leu Phe Ile Ala Lys Glu Thr Thr Leu Phe Leu Ala Ala ThrAsn 275 280 285 Ile Cys Met Asp Pro Leu Ile Tyr Ile Phe Leu Cys Lys LysPhe Thr 290 295 300 Glu Lys Leu Pro Cys Met Gln Gly Arg Lys Thr Thr AlaSer Ser Gln 305 310 315 320 Glu Asn His Ser Ser Gln Thr Asp Asn Ile ThrLeu Gly 325 330 21 1122 DNA Homo sapiens 21 atggccaaca ctaccggagagcctgaggag gtgagcggcg ctctgtcccc accgtccgca 60 tcagcttatg tgaagctggtactgctggga ctgattatgt gcgtgagcct ggcgggtaac 120 gccatcttgt ccctgctggtgctcaaggag cgtgccctgc acaaggctcc ttactacttc 180 ctgctggacc tgtgcctggccgatggcata cgctctgccg tctgcttccc ctttgtgctg 240 gcttctgtgc gccacggctcttcatggacc ttcagtgcac tcagctgcaa gattgtggcc 300 tttatggccg tgctcttttgcttccatgcg gccttcatgc tgttctgcat cagcgtcacc 360 cgctacatgg ccatcgcccaccaccgcttc tacgccaagc gcatgacact ctggacatgc 420 gcggctgtca tctgcatggcctggaccctg tctgtggcca tggccttccc acctgtcttt 480 gacgtgggca cctacaagtttattcgggag gaggaccagt gcatctttga gcatcgctac 540 ttcaaggcca atgacacgctgggcttcatg cttatgttgg ctgtgctcat ggcagctacc 600 catgctgtct acggcaagctgctcctcttc gagtatcgtc accgcaagat gaagccagtg 660 cagatggtgc cagccatcagccagaactgg acattccatg gtcccggggc caccggccag 720 gctgctgcca actggatcgccggctttggc cgtgggccca tgccaccaac cctgctgggt 780 atccggcaga atgggcatgcagccagccgg cggctactgg gcatggacga ggtcaagggt 840 gaaaagcagc tgggccgcatgttctacgcg atcacactgc tctttctgct cctctggtca 900 ccctacatcg tggcctgctactggcgagtg tttgtgaaag cctgtgctgt gccccaccgc 960 tacctggcca ctgctgtttggatgagcttc gcccaggctg ccgtcaaccc aattgtctgc 1020 ttcctgctca acaaggacctcaagaagtgc ctgaccactc acgccccctg ctggggcaca 1080 ggaggtgccc cggctcccagagaaccctac tgtgtcatgt ga 1122 22 373 PRT Homo sapiens 22 Met Ala Asn ThrThr Gly Glu Pro Glu Glu Val Ser Gly Ala Leu Ser 1 5 10 15 Pro Pro SerAla Ser Ala Tyr Val Lys Leu Val Leu Leu Gly Leu Ile 20 25 30 Met Cys ValSer Leu Ala Gly Asn Ala Ile Leu Ser Leu Leu Val Leu 35 40 45 Lys Glu ArgAla Leu His Lys Ala Pro Tyr Tyr Phe Leu Leu Asp Leu 50 55 60 Cys Leu AlaAsp Gly Ile Arg Ser Ala Val Cys Phe Pro Phe Val Leu 65 70 75 80 Ala SerVal Arg His Gly Ser Ser Trp Thr Phe Ser Ala Leu Ser Cys 85 90 95 Lys IleVal Ala Phe Met Ala Val Leu Phe Cys Phe His Ala Ala Phe 100 105 110 MetLeu Phe Cys Ile Ser Val Thr Arg Tyr Met Ala Ile Ala His His 115 120 125Arg Phe Tyr Ala Lys Arg Met Thr Leu Trp Thr Cys Ala Ala Val Ile 130 135140 Cys Met Ala Trp Thr Leu Ser Val Ala Met Ala Phe Pro Pro Val Phe 145150 155 160 Asp Val Gly Thr Tyr Lys Phe Ile Arg Glu Glu Asp Gln Cys IlePhe 165 170 175 Glu His Arg Tyr Phe Lys Ala Asn Asp Thr Leu Gly Phe MetLeu Met 180 185 190 Leu Ala Val Leu Met Ala Ala Thr His Ala Val Tyr GlyLys Leu Leu 195 200 205 Leu Phe Glu Tyr Arg His Arg Lys Met Lys Pro ValGln Met Val Pro 210 215 220 Ala Ile Ser Gln Asn Trp Thr Phe His Gly ProGly Ala Thr Gly Gln 225 230 235 240 Ala Ala Ala Asn Trp Ile Ala Gly PheGly Arg Gly Pro Met Pro Pro 245 250 255 Thr Leu Leu Gly Ile Arg Gln AsnGly His Ala Ala Ser Arg Arg Leu 260 265 270 Leu Gly Met Asp Glu Val LysGly Glu Lys Gln Leu Gly Arg Met Phe 275 280 285 Tyr Ala Ile Thr Leu LeuPhe Leu Leu Leu Trp Ser Pro Tyr Ile Val 290 295 300 Ala Cys Tyr Trp ArgVal Phe Val Lys Ala Cys Ala Val Pro His Arg 305 310 315 320 Tyr Leu AlaThr Ala Val Trp Met Ser Phe Ala Gln Ala Ala Val Asn 325 330 335 Pro IleVal Cys Phe Leu Leu Asn Lys Asp Leu Lys Lys Cys Leu Thr 340 345 350 ThrHis Ala Pro Cys Trp Gly Thr Gly Gly Ala Pro Ala Pro Arg Glu 355 360 365Pro Tyr Cys Val Met 370 23 1053 DNA Homo sapiens 23 atggctttggaacagaacca gtcaacagat tattattatg aggaaaatga aatgaatggc 60 acttatgactacagtcaata tgaattgatc tgtatcaaag aagatgtcag agaatttgca 120 aaagttttcctccctgtatt cctcacaata gctttcgtca ttggacttgc aggcaattcc 180 atggtagtggcaatttatgc ctattacaag aaacagagaa ccaaaacaga tgtgtacatc 240 ctgaatttggctgtagcaga tttactcctt ctattcactc tgcctttttg ggctgttaat 300 gcagttcatgggtgggtttt agggaaaata atgtgcaaaa taacttcagc cttgtacaca 360 ctaaactttgtctctggaat gcagtttctg gcttgcatca gcatagacag atatgtggca 420 gtaactaatgtccccagcca atcaggagtg ggaaaaccat gctggatcat ctgtttctgt 480 gtctggatggctgccatctt gctgagcata ccccagctgg ttttttatac agtaaatgac 540 aatgctaggtgcattcccat tttcccccgc tacctaggaa catcaatgaa agcattgatt 600 caaatgctagagatctgcat tggatttgta gtaccctttc ttattatggg ggtgtgctac 660 tttatcacggcaaggacact catgaagatg ccaaacatta aaatatctcg acccctaaaa 720 gttctgctcacagtcgttat agttttcatt gtcactcaac tgccttataa cattgtcaag 780 ttctgccgagccatagacat catctactcc ctgatcacca gctgcaacat gagcaaacgc 840 atggacatcgccatccaagt cacagaaagc attgcactct ttcacagctg cctcaaccca 900 atcctttatgtttttatggg agcatctttc aaaaactacg ttatgaaagt ggccaagaaa 960 tatgggtcctggagaagaca gagacaaagt gtggaggagt ttccttttga ttctgagggt 1020 cctacagagccaaccagtac ttttagcatt taa 1053 24 350 PRT Homo sapiens 24 Met Ala LeuGlu Gln Asn Gln Ser Thr Asp Tyr Tyr Tyr Glu Glu Asn 1 5 10 15 Glu MetAsn Gly Thr Tyr Asp Tyr Ser Gln Tyr Glu Leu Ile Cys Ile 20 25 30 Lys GluAsp Val Arg Glu Phe Ala Lys Val Phe Leu Pro Val Phe Leu 35 40 45 Thr IleAla Phe Val Ile Gly Leu Ala Gly Asn Ser Met Val Val Ala 50 55 60 Ile TyrAla Tyr Tyr Lys Lys Gln Arg Thr Lys Thr Asp Val Tyr Ile 65 70 75 80 LeuAsn Leu Ala Val Ala Asp Leu Leu Leu Leu Phe Thr Leu Pro Phe 85 90 95 TrpAla Val Asn Ala Val His Gly Trp Val Leu Gly Lys Ile Met Cys 100 105 110Lys Ile Thr Ser Ala Leu Tyr Thr Leu Asn Phe Val Ser Gly Met Gln 115 120125 Phe Leu Ala Cys Ile Ser Ile Asp Arg Tyr Val Ala Val Thr Asn Val 130135 140 Pro Ser Gln Ser Gly Val Gly Lys Pro Cys Trp Ile Ile Cys Phe Cys145 150 155 160 Val Trp Met Ala Ala Ile Leu Leu Ser Ile Pro Gln Leu ValPhe Tyr 165 170 175 Thr Val Asn Asp Asn Ala Arg Cys Ile Pro Ile Phe ProArg Tyr Leu 180 185 190 Gly Thr Ser Met Lys Ala Leu Ile Gln Met Leu GluIle Cys Ile Gly 195 200 205 Phe Val Val Pro Phe Leu Ile Met Gly Val CysTyr Phe Ile Thr Ala 210 215 220 Arg Thr Leu Met Lys Met Pro Asn Ile LysIle Ser Arg Pro Leu Lys 225 230 235 240 Val Leu Leu Thr Val Val Ile ValPhe Ile Val Thr Gln Leu Pro Tyr 245 250 255 Asn Ile Val Lys Phe Cys ArgAla Ile Asp Ile Ile Tyr Ser Leu Ile 260 265 270 Thr Ser Cys Asn Met SerLys Arg Met Asp Ile Ala Ile Gln Val Thr 275 280 285 Glu Ser Ile Ala LeuPhe His Ser Cys Leu Asn Pro Ile Leu Tyr Val 290 295 300 Phe Met Gly AlaSer Phe Lys Asn Tyr Val Met Lys Val Ala Lys Lys 305 310 315 320 Tyr GlySer Trp Arg Arg Gln Arg Gln Ser Val Glu Glu Phe Pro Phe 325 330 335 AspSer Glu Gly Pro Thr Glu Pro Thr Ser Thr Phe Ser Ile 340 345 350 25 1116DNA Homo sapiens 25 atgccaggaa acgccacccc agtgaccacc actgccccgtgggcctccct gggcctctcc 60 gccaagacct gcaacaacgt gtccttcgaa gagagcaggatagtcctggt cgtggtgtac 120 agcgcggtgt gcacgctggg ggtgccggcc aactgcctgactgcgtggct ggcgctgctg 180 caggtactgc agggcaacgt gctggccgtc tacctgctctgcctggcact ctgcgaactg 240 ctgtacacag gcacgctgcc actctgggtc atctatatccgcaaccagca ccgctggacc 300 ctaggcctgc tggcctcgaa ggtgaccgcc tacatcttcttctgcaacat ctacgtcagc 360 atcctcttcc tgtgctgcat ctcctgcgac cgcttcgtggccgtggtgta cgcgctggag 420 agtcggggcc gccgccgccg gaggaccgcc atcctcatctccgcctgcat cttcatcctc 480 gtcgggatcg ttcactaccc ggtgttccag acggaagacaaggagacctg ctttgacatg 540 ctgcagatgg acagcaggat tgccgggtac tactacgccaggttcaccgt tggctttgcc 600 atccctctct ccatcatcgc cttcaccaac caccggattttcaggagcat caagcagagc 660 atgggcttaa gcgctgccca gaaggccaag gtgaagcactcggccatcgc ggtggttgtc 720 atcttcctag tctgcttcgc cccgtaccac ctggttctcctcgtcaaagc cgctgccttt 780 tcctactaca gaggagacag gaacgccatg tgcggcttggaggaaaggct gtacacagcc 840 tctgtggtgt ttctgtgcct gtccacggtg aacggcgtggctgaccccat tatctacgtg 900 ctggccacgg accattcccg ccaagaagtg tccagaatccataaggggtg gaaagagtgg 960 tccatgaaga cagacgtcac caggctcacc cacagcagggacaccgagga gctgcagtcg 1020 cccgtggccc ttgcagacca ctacaccttc tccaggcccgtgcacccacc agggtcacca 1080 tgccctgcaa agaggctgat tgaggagtcc tgctga 111626 371 PRT Homo sapiens 26 Met Pro Gly Asn Ala Thr Pro Val Thr Thr ThrAla Pro Trp Ala Ser 1 5 10 15 Leu Gly Leu Ser Ala Lys Thr Cys Asn AsnVal Ser Phe Glu Glu Ser 20 25 30 Arg Ile Val Leu Val Val Val Tyr Ser AlaVal Cys Thr Leu Gly Val 35 40 45 Pro Ala Asn Cys Leu Thr Ala Trp Leu AlaLeu Leu Gln Val Leu Gln 50 55 60 Gly Asn Val Leu Ala Val Tyr Leu Leu CysLeu Ala Leu Cys Glu Leu 65 70 75 80 Leu Tyr Thr Gly Thr Leu Pro Leu TrpVal Ile Tyr Ile Arg Asn Gln 85 90 95 His Arg Trp Thr Leu Gly Leu Leu AlaSer Lys Val Thr Ala Tyr Ile 100 105 110 Phe Phe Cys Asn Ile Tyr Val SerIle Leu Phe Leu Cys Cys Ile Ser 115 120 125 Cys Asp Arg Phe Val Ala ValVal Tyr Ala Leu Glu Ser Arg Gly Arg 130 135 140 Arg Arg Arg Arg Thr AlaIle Leu Ile Ser Ala Cys Ile Phe Ile Leu 145 150 155 160 Val Gly Ile ValHis Tyr Pro Val Phe Gln Thr Glu Asp Lys Glu Thr 165 170 175 Cys Phe AspMet Leu Gln Met Asp Ser Arg Ile Ala Gly Tyr Tyr Tyr 180 185 190 Ala ArgPhe Thr Val Gly Phe Ala Ile Pro Leu Ser Ile Ile Ala Phe 195 200 205 ThrAsn His Arg Ile Phe Arg Ser Ile Lys Gln Ser Met Gly Leu Ser 210 215 220Ala Ala Gln Lys Ala Lys Val Lys His Ser Ala Ile Ala Val Val Val 225 230235 240 Ile Phe Leu Val Cys Phe Ala Pro Tyr His Leu Val Leu Leu Val Lys245 250 255 Ala Ala Ala Phe Ser Tyr Tyr Arg Gly Asp Arg Asn Ala Met CysGly 260 265 270 Leu Glu Glu Arg Leu Tyr Thr Ala Ser Val Val Phe Leu CysLeu Ser 275 280 285 Thr Val Asn Gly Val Ala Asp Pro Ile Ile Tyr Val LeuAla Thr Asp 290 295 300 His Ser Arg Gln Glu Val Ser Arg Ile His Lys GlyTrp Lys Glu Trp 305 310 315 320 Ser Met Lys Thr Asp Val Thr Arg Leu ThrHis Ser Arg Asp Thr Glu 325 330 335 Glu Leu Gln Ser Pro Val Ala Leu AlaAsp His Tyr Thr Phe Ser Arg 340 345 350 Pro Val His Pro Pro Gly Ser ProCys Pro Ala Lys Arg Leu Ile Glu 355 360 365 Glu Ser Cys 370 27 1113 DNAHomo sapiens 27 atggcgaact atagccatgc agctgacaac attttgcaaa atctctcgcctctaacagcc 60 tttctgaaac tgacttcctt gggtttcata ataggagtca gcgtggtgggcaacctcctg 120 atctccattt tgctagtgaa agataagacc ttgcatagag caccttactacttcctgttg 180 gatctttgct gttcagatat cctcagatct gcaatttgtt tcccatttgtgttcaactct 240 gtcaaaaatg gctctacctg gacttatggg actctgactt gcaaagtgattgcctttctg 300 ggggttttgt cctgtttcca cactgctttc atgctcttct gcatcagtgtcaccagatac 360 ttagctatcg cccatcaccg cttctataca aagaggctga ccttttggacgtgtctggct 420 gtgatctgta tggtgtggac tctgtctgtg gccatggcat ttcccccggttttagacgtg 480 ggcacttact cattcattag ggaggaagat caatgcacct tccaacaccgctccttcagg 540 gctaatgatt ccttaggatt tatgctgctt cttgctctca tcctcctagccacacagctt 600 gtctacctca agctgatatt tttcgtccac gatcgaagaa aaatgaagccagtccagttt 660 gtagcagcag tcagccagaa ctggactttt catggtcctg gagccagtggccaggcagct 720 gccaattggc tagcaggatt tggaaggggt cccacaccac ccaccttgctgggcatcagg 780 caaaatgcaa acaccacagg cagaagaagg ctattggtct tagacgagttcaaaatggag 840 aaaagaatca gcagaatgtt ctatataatg acttttctgt ttctaaccttgtggggcccc 900 tacctggtgg cctgttattg gagagttttt gcaagagggc ctgtagtaccagggggattt 960 ctaacagctg ctgtctggat gagttttgcc caagcaggaa tcaatccttttgtctgcatt 1020 ttctcaaaca gggagctgag gcgctgtttc agcacaaccc ttctttactgcagaaaatcc 1080 aggttaccaa gggaacctta ctgtgttata tga 1113 28 370 PRTHomo sapiens 28 Met Ala Asn Tyr Ser His Ala Ala Asp Asn Ile Leu Gln AsnLeu Ser 1 5 10 15 Pro Leu Thr Ala Phe Leu Lys Leu Thr Ser Leu Gly PheIle Ile Gly 20 25 30 Val Ser Val Val Gly Asn Leu Leu Ile Ser Ile Leu LeuVal Lys Asp 35 40 45 Lys Thr Leu His Arg Ala Pro Tyr Tyr Phe Leu Leu AspLeu Cys Cys 50 55 60 Ser Asp Ile Leu Arg Ser Ala Ile Cys Phe Pro Phe ValPhe Asn Ser 65 70 75 80 Val Lys Asn Gly Ser Thr Trp Thr Tyr Gly Thr LeuThr Cys Lys Val 85 90 95 Ile Ala Phe Leu Gly Val Leu Ser Cys Phe His ThrAla Phe Met Leu 100 105 110 Phe Cys Ile Ser Val Thr Arg Tyr Leu Ala IleAla His His Arg Phe 115 120 125 Tyr Thr Lys Arg Leu Thr Phe Trp Thr CysLeu Ala Val Ile Cys Met 130 135 140 Val Trp Thr Leu Ser Val Ala Met AlaPhe Pro Pro Val Leu Asp Val 145 150 155 160 Gly Thr Tyr Ser Phe Ile ArgGlu Glu Asp Gln Cys Thr Phe Gln His 165 170 175 Arg Ser Phe Arg Ala AsnAsp Ser Leu Gly Phe Met Leu Leu Leu Ala 180 185 190 Leu Ile Leu Leu AlaThr Gln Leu Val Tyr Leu Lys Leu Ile Phe Phe 195 200 205 Val His Asp ArgArg Lys Met Lys Pro Val Gln Phe Val Ala Ala Val 210 215 220 Ser Gln AsnTrp Thr Phe His Gly Pro Gly Ala Ser Gly Gln Ala Ala 225 230 235 240 AlaAsn Trp Leu Ala Gly Phe Gly Arg Gly Pro Thr Pro Pro Thr Leu 245 250 255Leu Gly Ile Arg Gln Asn Ala Asn Thr Thr Gly Arg Arg Arg Leu Leu 260 265270 Val Leu Asp Glu Phe Lys Met Glu Lys Arg Ile Ser Arg Met Phe Tyr 275280 285 Ile Met Thr Phe Leu Phe Leu Thr Leu Trp Gly Pro Tyr Leu Val Ala290 295 300 Cys Tyr Trp Arg Val Phe Ala Arg Gly Pro Val Val Pro Gly GlyPhe 305 310 315 320 Leu Thr Ala Ala Val Trp Met Ser Phe Ala Gln Ala GlyIle Asn Pro 325 330 335 Phe Val Cys Ile Phe Ser Asn Arg Glu Leu Arg ArgCys Phe Ser Thr 340 345 350 Thr Leu Leu Tyr Cys Arg Lys Ser Arg Leu ProArg Glu Pro Tyr Cys 355 360 365 Val Ile 370 29 1080 DNA Homo sapiens 29atgcaggtcc cgaacagcac cggcccggac aacgcgacgc tgcagatgct gcggaacccg 60gcgatcgcgg tggccctgcc cgtggtgtac tcgctggtgg cggcggtcag catcccgggc 120aacctcttct ctctgtgggt gctgtgccgg cgcatggggc ccagatcccc gtcggtcatc 180ttcatgatca acctgagcgt cacggacctg atgctggcca gcgtgttgcc tttccaaatc 240tactaccatt gcaaccgcca ccactgggta ttcggggtgc tgctttgcaa cgtggtgacc 300gtggcctttt acgcaaacat gtattccagc atcctcacca tgacctgtat cagcgtggag 360cgcttcctgg gggtcctgta cccgctcagc tccaagcgct ggcgccgccg tcgttacgcg 420gtggccgcgt gtgcagggac ctggctgctg ctcctgaccg ccctgtgccc gctggcgcgc 480accgatctca cctacccggt gcacgccctg ggcatcatca cctgcttcga cgtcctcaag 540tggacgatgc tccccagcgt ggccatgtgg gccgtgttcc tcttcaccat cttcatcctg 600ctgttcctca tcccgttcgt gatcaccgtg gcttgttaca cggccaccat cctcaagctg 660ttgcgcacgg aggaggcgca cggccgggag cagcggaggc gcgcggtggg cctggccgcg 720gtggtcttgc tggcctttgt cacctgcttc gcccccaaca acttcgtgct cctggcgcac 780atcgtgagcc gcctgttcta cggcaagagc tactaccacg tgtacaagct cacgctgtgt 840ctcagctgcc tcaacaactg tctggacccg tttgtttatt actttgcgtc ccgggaattc 900cagctgcgcc tgcgggaata tttgggctgc cgccgggtgc ccagagacac cctggacacg 960cgccgcgaga gcctcttctc cgccaggacc acgtccgtgc gctccgaggc cggtgcgcac 1020cctgaaggga tggagggagc caccaggccc ggcctccaga ggcaggagag tgtgttctga 108030 359 PRT Homo sapiens 30 Met Gln Val Pro Asn Ser Thr Gly Pro Asp AsnAla Thr Leu Gln Met 1 5 10 15 Leu Arg Asn Pro Ala Ile Ala Val Ala LeuPro Val Val Tyr Ser Leu 20 25 30 Val Ala Ala Val Ser Ile Pro Gly Asn LeuPhe Ser Leu Trp Val Leu 35 40 45 Cys Arg Arg Met Gly Pro Arg Ser Pro SerVal Ile Phe Met Ile Asn 50 55 60 Leu Ser Val Thr Asp Leu Met Leu Ala SerVal Leu Pro Phe Gln Ile 65 70 75 80 Tyr Tyr His Cys Asn Arg His His TrpVal Phe Gly Val Leu Leu Cys 85 90 95 Asn Val Val Thr Val Ala Phe Tyr AlaAsn Met Tyr Ser Ser Ile Leu 100 105 110 Thr Met Thr Cys Ile Ser Val GluArg Phe Leu Gly Val Leu Tyr Pro 115 120 125 Leu Ser Ser Lys Arg Trp ArgArg Arg Arg Tyr Ala Val Ala Ala Cys 130 135 140 Ala Gly Thr Trp Leu LeuLeu Leu Thr Ala Leu Cys Pro Leu Ala Arg 145 150 155 160 Thr Asp Leu ThrTyr Pro Val His Ala Leu Gly Ile Ile Thr Cys Phe 165 170 175 Asp Val LeuLys Trp Thr Met Leu Pro Ser Val Ala Met Trp Ala Val 180 185 190 Phe LeuPhe Thr Ile Phe Ile Leu Leu Phe Leu Ile Pro Phe Val Ile 195 200 205 ThrVal Ala Cys Tyr Thr Ala Thr Ile Leu Lys Leu Leu Arg Thr Glu 210 215 220Glu Ala His Gly Arg Glu Gln Arg Arg Arg Ala Val Gly Leu Ala Ala 225 230235 240 Val Val Leu Leu Ala Phe Val Thr Cys Phe Ala Pro Asn Asn Phe Val245 250 255 Leu Leu Ala His Ile Val Ser Arg Leu Phe Tyr Gly Lys Ser TyrTyr 260 265 270 His Val Tyr Lys Leu Thr Leu Cys Leu Ser Cys Leu Asn AsnCys Leu 275 280 285 Asp Pro Phe Val Tyr Tyr Phe Ala Ser Arg Glu Phe GlnLeu Arg Leu 290 295 300 Arg Glu Tyr Leu Gly Cys Arg Arg Val Pro Arg AspThr Leu Asp Thr 305 310 315 320 Arg Arg Glu Ser Leu Phe Ser Ala Arg ThrThr Ser Val Arg Ser Glu 325 330 335 Ala Gly Ala His Pro Glu Gly Met GluGly Ala Thr Arg Pro Gly Leu 340 345 350 Gln Arg Gln Glu Ser Val Phe 35531 1503 DNA Homo sapiens 31 atggagcgtc cctgggagga cagcccaggc ccggagggggcagctgaggg ctcgcctgtg 60 ccagtcgccg ccggggcgcg ctccggtgcc gcggcgagtggcacaggctg gcagccatgg 120 gctgagtgcc cgggacccaa ggggaggggg caactgctggcgaccgccgg ccctttgcgt 180 cgctggcccg ccccctcgcc tgccagctcc agccccgcccccggagcggc gtccgctcac 240 tcggttcaag gcagcgcgac tgcgggtggc gcacgaccagggcgcagacc ttggggcgcg 300 cggcccatgg agtcggggct gctgcggccg gcgccggtgagcgaggtcat cgtcctgcat 360 tacaactaca ccggcaagct ccgcggtgcg agctaccagccgggtgccgg cctgcgcgcc 420 gacgccgtgg tgtgcctggc ggtgtgcgcc ttcatcgtgctagagaatct agccgtgttg 480 ttggtgctcg gacgccaccc gcgcttccac gctcccatgttcctgctcct gggcagcctc 540 acgttgtcgg atctgctggc aggcgccgcc tacgccgccaacatcctact gtcggggccg 600 ctcacgctga aactgtcccc cgcgctctgg ttcgcacgggagggaggcgt cttcgtggca 660 ctcactgcgt ccgtgctgag cctcctggcc atcgcgctggagcgcagcct caccatggcg 720 cgcagggggc ccgcgcccgt ctccagtcgg gggcgcacgctggcgatggc agccgcggcc 780 tggggcgtgt cgctgctcct cgggctcctg ccagcgctgggctggaattg cctgggtcgc 840 ctggacgctt gctccactgt cttgccgctc tacgccaaggcctacgtgct cttctgcgtg 900 ctcgccttcg tgggcatcct ggccgcgatc tgtgcactctacgcgcgcat ctactgccag 960 gtacgcgcca acgcgcggcg cctgccggca cggcccgggactgcggggac cacctcgacc 1020 cgggcgcgtc gcaagccgcg ctctctggcc ttgctgcgcacgctcagcgt ggtgctcctg 1080 gcctttgtgg catgttgggg ccccctcttc ctgctgctgttgctcgacgt ggcgtgcccg 1140 gcgcgcacct gtcctgtact cctgcaggcc gatcccttcctgggactggc catggccaac 1200 tcacttctga accccatcat ctacacgctc accaaccgcgacctgcgcca cgcgctcctg 1260 cgcctggtct gctgcggacg ccactcctgc ggcagagacccgagtggctc ccagcagtcg 1320 gcgagcgcgg ctgaggcttc cgggggcctg cgccgctgcctgcccccggg ccttgatggg 1380 agcttcagcg gctcggagcg ctcatcgccc cagcgcgacgggctggacac cagcggctcc 1440 acaggcagcc ccggtgcacc cacagccgcc cggactctggtatcagaacc ggctgcagac 1500 tga 1503 32 500 PRT Homo sapiens 32 Met GluArg Pro Trp Glu Asp Ser Pro Gly Pro Glu Gly Ala Ala Glu 1 5 10 15 GlySer Pro Val Pro Val Ala Ala Gly Ala Arg Ser Gly Ala Ala Ala 20 25 30 SerGly Thr Gly Trp Gln Pro Trp Ala Glu Cys Pro Gly Pro Lys Gly 35 40 45 ArgGly Gln Leu Leu Ala Thr Ala Gly Pro Leu Arg Arg Trp Pro Ala 50 55 60 ProSer Pro Ala Ser Ser Ser Pro Ala Pro Gly Ala Ala Ser Ala His 65 70 75 80Ser Val Gln Gly Ser Ala Thr Ala Gly Gly Ala Arg Pro Gly Arg Arg 85 90 95Pro Trp Gly Ala Arg Pro Met Glu Ser Gly Leu Leu Arg Pro Ala Pro 100 105110 Val Ser Glu Val Ile Val Leu His Tyr Asn Tyr Thr Gly Lys Leu Arg 115120 125 Gly Ala Ser Tyr Gln Pro Gly Ala Gly Leu Arg Ala Asp Ala Val Val130 135 140 Cys Leu Ala Val Cys Ala Phe Ile Val Leu Glu Asn Leu Ala ValLeu 145 150 155 160 Leu Val Leu Gly Arg His Pro Arg Phe His Ala Pro MetPhe Leu Leu 165 170 175 Leu Gly Ser Leu Thr Leu Ser Asp Leu Leu Ala GlyAla Ala Tyr Ala 180 185 190 Ala Asn Ile Leu Leu Ser Gly Pro Leu Thr LeuLys Leu Ser Pro Ala 195 200 205 Leu Trp Phe Ala Arg Glu Gly Gly Val PheVal Ala Leu Thr Ala Ser 210 215 220 Val Leu Ser Leu Leu Ala Ile Ala LeuGlu Arg Ser Leu Thr Met Ala 225 230 235 240 Arg Arg Gly Pro Ala Pro ValSer Ser Arg Gly Arg Thr Leu Ala Met 245 250 255 Ala Ala Ala Ala Trp GlyVal Ser Leu Leu Leu Gly Leu Leu Pro Ala 260 265 270 Leu Gly Trp Asn CysLeu Gly Arg Leu Asp Ala Cys Ser Thr Val Leu 275 280 285 Pro Leu Tyr AlaLys Ala Tyr Val Leu Phe Cys Val Leu Ala Phe Val 290 295 300 Gly Ile LeuAla Ala Ile Cys Ala Leu Tyr Ala Arg Ile Tyr Cys Gln 305 310 315 320 ValArg Ala Asn Ala Arg Arg Leu Pro Ala Arg Pro Gly Thr Ala Gly 325 330 335Thr Thr Ser Thr Arg Ala Arg Arg Lys Pro Arg Ser Leu Ala Leu Leu 340 345350 Arg Thr Leu Ser Val Val Leu Leu Ala Phe Val Ala Cys Trp Gly Pro 355360 365 Leu Phe Leu Leu Leu Leu Leu Asp Val Ala Cys Pro Ala Arg Thr Cys370 375 380 Pro Val Leu Leu Gln Ala Asp Pro Phe Leu Gly Leu Ala Met AlaAsn 385 390 395 400 Ser Leu Leu Asn Pro Ile Ile Tyr Thr Leu Thr Asn ArgAsp Leu Arg 405 410 415 His Ala Leu Leu Arg Leu Val Cys Cys Gly Arg HisSer Cys Gly Arg 420 425 430 Asp Pro Ser Gly Ser Gln Gln Ser Ala Ser AlaAla Glu Ala Ser Gly 435 440 445 Gly Leu Arg Arg Cys Leu Pro Pro Gly LeuAsp Gly Ser Phe Ser Gly 450 455 460 Ser Glu Arg Ser Ser Pro Gln Arg AspGly Leu Asp Thr Ser Gly Ser 465 470 475 480 Thr Gly Ser Pro Gly Ala ProThr Ala Ala Arg Thr Leu Val Ser Glu 485 490 495 Pro Ala Ala Asp 500 331029 DNA Homo sapiens 33 atgcaagccg tcgacaatct cacctctgcg cctgggaacaccagtctgtg caccagagac 60 tacaaaatca cccaggtcct cttcccactg ctctacactgtcctgttttt tgttggactt 120 atcacaaatg gcctggcgat gaggattttc tttcaaatccggagtaaatc aaactttatt 180 atttttctta agaacacagt catttctgat cttctcatgattctgacttt tccattcaaa 240 attcttagtg atgccaaact gggaacagga ccactgagaacttttgtgtg tcaagttacc 300 tccgtcatat tttatttcac aatgtatatc agtatttcattcctgggact gataactatc 360 gatcgctacc agaagaccac caggccattt aaaacatccaaccccaaaaa tctcttgggg 420 gctaagattc tctctgttgt catctgggca ttcatgttcttactctcttt gcctaacatg 480 attctgacca acaggcagcc gagagacaag aatgtgaagaaatgctcttt ccttaaatca 540 gagttcggtc tagtctggca tgaaatagta aattacatctgtcaagtcat tttctggatt 600 aatttcttaa ttgttattgt atgttataca ctcattacaaaagaactgta ccggtcatac 660 gtaagaacga ggggtgtagg taaagtcccc aggaaaaaggtgaacgtcaa agttttcatt 720 atcattgctg tattctttat ttgttttgtt cctttccattttgcccgaat tccttacacc 780 ctgagccaaa cccgggatgt ctttgactgc actgctgaaaatactctgtt ctatgtgaaa 840 gagagcactc tgtggttaac ttccttaaat gcatgcctggatccgttcat ctattttttc 900 ctttgcaagt ccttcagaaa ttccttgata agtatgctgaagtgccccaa ttctgcaaca 960 tctctgtccc aggacaatag gaaaaaagaa caggatggtggtgacccaaa tgaagagact 1020 ccaatgtaa 1029 34 342 PRT Homo sapiens 34 MetGln Ala Val Asp Asn Leu Thr Ser Ala Pro Gly Asn Thr Ser Leu 1 5 10 15Cys Thr Arg Asp Tyr Lys Ile Thr Gln Val Leu Phe Pro Leu Leu Tyr 20 25 30Thr Val Leu Phe Phe Val Gly Leu Ile Thr Asn Gly Leu Ala Met Arg 35 40 45Ile Phe Phe Gln Ile Arg Ser Lys Ser Asn Phe Ile Ile Phe Leu Lys 50 55 60Asn Thr Val Ile Ser Asp Leu Leu Met Ile Leu Thr Phe Pro Phe Lys 65 70 7580 Ile Leu Ser Asp Ala Lys Leu Gly Thr Gly Pro Leu Arg Thr Phe Val 85 9095 Cys Gln Val Thr Ser Val Ile Phe Tyr Phe Thr Met Tyr Ile Ser Ile 100105 110 Ser Phe Leu Gly Leu Ile Thr Ile Asp Arg Tyr Gln Lys Thr Thr Arg115 120 125 Pro Phe Lys Thr Ser Asn Pro Lys Asn Leu Leu Gly Ala Lys IleLeu 130 135 140 Ser Val Val Ile Trp Ala Phe Met Phe Leu Leu Ser Leu ProAsn Met 145 150 155 160 Ile Leu Thr Asn Arg Gln Pro Arg Asp Lys Asn ValLys Lys Cys Ser 165 170 175 Phe Leu Lys Ser Glu Phe Gly Leu Val Trp HisGlu Ile Val Asn Tyr 180 185 190 Ile Cys Gln Val Ile Phe Trp Ile Asn PheLeu Ile Val Ile Val Cys 195 200 205 Tyr Thr Leu Ile Thr Lys Glu Leu TyrArg Ser Tyr Val Arg Thr Arg 210 215 220 Gly Val Gly Lys Val Pro Arg LysLys Val Asn Val Lys Val Phe Ile 225 230 235 240 Ile Ile Ala Val Phe PheIle Cys Phe Val Pro Phe His Phe Ala Arg 245 250 255 Ile Pro Tyr Thr LeuSer Gln Thr Arg Asp Val Phe Asp Cys Thr Ala 260 265 270 Glu Asn Thr LeuPhe Tyr Val Lys Glu Ser Thr Leu Trp Leu Thr Ser 275 280 285 Leu Asn AlaCys Leu Asp Pro Phe Ile Tyr Phe Phe Leu Cys Lys Ser 290 295 300 Phe ArgAsn Ser Leu Ile Ser Met Leu Lys Cys Pro Asn Ser Ala Thr 305 310 315 320Ser Leu Ser Gln Asp Asn Arg Lys Lys Glu Gln Asp Gly Gly Asp Pro 325 330335 Asn Glu Glu Thr Pro Met 340 35 1077 DNA Homo sapiens 35 atgtcggtctgctaccgtcc cccagggaac gagacactgc tgagctggaa gacttcgcgg 60 gccacaggcacagccttcct gctgctggcg gcgctgctgg ggctgcctgg caacggcttc 120 gtggtgtggagcttggcggg ctggcggcct gcacgggggc gaccgctggc ggccacgctt 180 gtgctgcacctggcgctggc cgacggcgcg gtgctgctgc tcacgccgct ctttgtggcc 240 ttcctgacccggcaggcctg gccgctgggc caggcgggct gcaaggcggt gtactacgtg 300 tgcgcgctcagcatgtacgc cagcgtgctg ctcaccggcc tgctcagcct gcagcgctgc 360 ctcgcagtcacccgcccctt cctggcgcct cggctgcgca gcccggccct ggcccgccgc 420 ctgctgctggcggtctggct ggccgccctg ttgctcgccg tcccggccgc cgtctaccgc 480 cacctgtggagggaccgcgt atgccagctg tgccacccgt cgccggtcca cgccgccgcc 540 cacctgagcctggagactct gaccgctttc gtgcttcctt tcgggctgat gctcggctgc 600 tacagcgtgacgctggcacg gctgcggggc gcccgctggg gctccgggcg gcacggggcg 660 cgggtgggccggctggtgag cgccatcgtg cttgccttcg gcttgctctg ggccccctac 720 cacgcagtcaaccttctgca ggcggtcgca gcgctggctc caccggaagg ggccttggcg 780 aagctgggcggagccggcca ggcggcgcga gcgggaacta cggccttggc cttcttcagt 840 tctagcgtcaacccggtgct ctacgtcttc accgctggag atctgctgcc ccgggcaggt 900 ccccgtttcctcacgcggct cttcgaaggc tctggggagg cccgaggggg cggccgctct 960 agggaagggaccatggagct ccgaactacc cctcagctga aagtggtggg gcagggccgc 1020 ggcaatggagacccgggggg tgggatggag aaggacggtc cggaatggga cctttga 1077 36 358 PRT Homosapiens 36 Met Ser Val Cys Tyr Arg Pro Pro Gly Asn Glu Thr Leu Leu SerTrp 1 5 10 15 Lys Thr Ser Arg Ala Thr Gly Thr Ala Phe Leu Leu Leu AlaAla Leu 20 25 30 Leu Gly Leu Pro Gly Asn Gly Phe Val Val Trp Ser Leu AlaGly Trp 35 40 45 Arg Pro Ala Arg Gly Arg Pro Leu Ala Ala Thr Leu Val LeuHis Leu 50 55 60 Ala Leu Ala Asp Gly Ala Val Leu Leu Leu Thr Pro Leu PheVal Ala 65 70 75 80 Phe Leu Thr Arg Gln Ala Trp Pro Leu Gly Gln Ala GlyCys Lys Ala 85 90 95 Val Tyr Tyr Val Cys Ala Leu Ser Met Tyr Ala Ser ValLeu Leu Thr 100 105 110 Gly Leu Leu Ser Leu Gln Arg Cys Leu Ala Val ThrArg Pro Phe Leu 115 120 125 Ala Pro Arg Leu Arg Ser Pro Ala Leu Ala ArgArg Leu Leu Leu Ala 130 135 140 Val Trp Leu Ala Ala Leu Leu Leu Ala ValPro Ala Ala Val Tyr Arg 145 150 155 160 His Leu Trp Arg Asp Arg Val CysGln Leu Cys His Pro Ser Pro Val 165 170 175 His Ala Ala Ala His Leu SerLeu Glu Thr Leu Thr Ala Phe Val Leu 180 185 190 Pro Phe Gly Leu Met LeuGly Cys Tyr Ser Val Thr Leu Ala Arg Leu 195 200 205 Arg Gly Ala Arg TrpGly Ser Gly Arg His Gly Ala Arg Val Gly Arg 210 215 220 Leu Val Ser AlaIle Val Leu Ala Phe Gly Leu Leu Trp Ala Pro Tyr 225 230 235 240 His AlaVal Asn Leu Leu Gln Ala Val Ala Ala Leu Ala Pro Pro Glu 245 250 255 GlyAla Leu Ala Lys Leu Gly Gly Ala Gly Gln Ala Ala Arg Ala Gly 260 265 270Thr Thr Ala Leu Ala Phe Phe Ser Ser Ser Val Asn Pro Val Leu Tyr 275 280285 Val Phe Thr Ala Gly Asp Leu Leu Pro Arg Ala Gly Pro Arg Phe Leu 290295 300 Thr Arg Leu Phe Glu Gly Ser Gly Glu Ala Arg Gly Gly Gly Arg Ser305 310 315 320 Arg Glu Gly Thr Met Glu Leu Arg Thr Thr Pro Gln Leu LysVal Val 325 330 335 Gly Gln Gly Arg Gly Asn Gly Asp Pro Gly Gly Gly MetGlu Lys Asp 340 345 350 Gly Pro Glu Trp Asp Leu 355 37 1005 DNA Homosapiens 37 atgctgggga tcatggcatg gaatgcaact tgcaaaaact ggctggcagcagaggctgcc 60 ctggaaaagt actacctttc cattttttat gggattgagt tcgttgtgggagtccttgga 120 aataccattg ttgtttacgg ctacatcttc tctctgaaga actggaacagcagtaatatt 180 tatctcttta acctctctgt ctctgactta gcttttctgt gcaccctccccatgctgata 240 aggagttatg ccaatggaaa ctggatatat ggagacgtgc tctgcataagcaaccgatat 300 gtgcttcatg ccaacctcta taccagcatt ctctttctca cttttatcagcatagatcga 360 tacttgataa ttaagtatcc tttccgagaa caccttctgc aaaagaaagagtttgctatt 420 ttaatctcct tggccatttg ggttttagta accttagagt tactacccatacttcccctt 480 ataaatcctg ttataactga caatggcacc acctgtaatg attttgcaagttctggagac 540 cccaactaca acctcattta cagcatgtgt ctaacactgt tggggttccttattcctctt 600 tttgtgatgt gtttctttta ttacaagatt gctctcttcc taaagcagaggaataggcag 660 gttgctactg ctctgcccct tgaaaagcct ctcaacttgg tcatcatggcagtggtaatc 720 ttctctgtgc tttttacacc ctatcacgtc atgcggaatg tgaggatcgcttcacgcctg 780 gggagttgga agcagtatca gtgcactcag gtcgtcatca actccttttacattgtgaca 840 cggcctttgg cctttctgaa cagtgtcatc aaccctgtct tctattttcttttgggagat 900 cacttcaggg acatgctgat gaatcaactg agacacaact tcaaatcccttacatccttt 960 agcagatggg ctcatgaact cctactttca ttcagagaaa agtga 1005 38334 PRT Homo sapiens 38 Met Leu Gly Ile Met Ala Trp Asn Ala Thr Cys LysAsn Trp Leu Ala 1 5 10 15 Ala Glu Ala Ala Leu Glu Lys Tyr Tyr Leu SerIle Phe Tyr Gly Ile 20 25 30 Glu Phe Val Val Gly Val Leu Gly Asn Thr IleVal Val Tyr Gly Tyr 35 40 45 Ile Phe Ser Leu Lys Asn Trp Asn Ser Ser AsnIle Tyr Leu Phe Asn 50 55 60 Leu Ser Val Ser Asp Leu Ala Phe Leu Cys ThrLeu Pro Met Leu Ile 65 70 75 80 Arg Ser Tyr Ala Asn Gly Asn Trp Ile TyrGly Asp Val Leu Cys Ile 85 90 95 Ser Asn Arg Tyr Val Leu His Ala Asn LeuTyr Thr Ser Ile Leu Phe 100 105 110 Leu Thr Phe Ile Ser Ile Asp Arg TyrLeu Ile Ile Lys Tyr Pro Phe 115 120 125 Arg Glu His Leu Leu Gln Lys LysGlu Phe Ala Ile Leu Ile Ser Leu 130 135 140 Ala Ile Trp Val Leu Val ThrLeu Glu Leu Leu Pro Ile Leu Pro Leu 145 150 155 160 Ile Asn Pro Val IleThr Asp Asn Gly Thr Thr Cys Asn Asp Phe Ala 165 170 175 Ser Ser Gly AspPro Asn Tyr Asn Leu Ile Tyr Ser Met Cys Leu Thr 180 185 190 Leu Leu GlyPhe Leu Ile Pro Leu Phe Val Met Cys Phe Phe Tyr Tyr 195 200 205 Lys IleAla Leu Phe Leu Lys Gln Arg Asn Arg Gln Val Ala Thr Ala 210 215 220 LeuPro Leu Glu Lys Pro Leu Asn Leu Val Ile Met Ala Val Val Ile 225 230 235240 Phe Ser Val Leu Phe Thr Pro Tyr His Val Met Arg Asn Val Arg Ile 245250 255 Ala Ser Arg Leu Gly Ser Trp Lys Gln Tyr Gln Cys Thr Gln Val Val260 265 270 Ile Asn Ser Phe Tyr Ile Val Thr Arg Pro Leu Ala Phe Leu AsnSer 275 280 285 Val Ile Asn Pro Val Phe Tyr Phe Leu Leu Gly Asp His PheArg Asp 290 295 300 Met Leu Met Asn Gln Leu Arg His Asn Phe Lys Ser LeuThr Ser Phe 305 310 315 320 Ser Arg Trp Ala His Glu Leu Leu Leu Ser PheArg Glu Lys 325 330 39 1296 DNA Homo sapiens 39 atgcaggcgc ttaacattaccccggagcag ttctctcggc tgctgcggga ccacaacctg 60 acgcgggagc agttcatcgctctgtaccgg ctgcgaccgc tcgtctacac cccagagctg 120 ccgggacgcg ccaagctggccctcgtgctc accggcgtgc tcatcttcgc cctggcgctc 180 tttggcaatg ctctggtgttctacgtggtg acccgcagca aggccatgcg caccgtcacc 240 aacatcttta tctgctccttggcgctcagt gacctgctca tcaccttctt ctgcattccc 300 gtcaccatgc tccagaacatttccgacaac tggctggggg gtgctttcat ttgcaagatg 360 gtgccatttg tccagtctaccgctgttgtg acagaaatgc tcactatgac ctgcattgct 420 gtggaaaggc accagggacttgtgcatcct tttaaaatga agtggcaata caccaaccga 480 agggctttca caatgctaggtgtggtctgg ctggtggcag tcatcgtagg atcacccatg 540 tggcacgtgc aacaacttgagatcaaatat gacttcctat atgaaaagga acacatctgc 600 tgcttagaag agtggaccagccctgtgcac cagaagatct acaccacctt catccttgtc 660 atcctcttcc tcctgcctcttatggtgatg cttattctgt acagtaaaat tggttatgaa 720 ctttggataa agaaaagagttggggatggt tcagtgcttc gaactattca tggaaaagaa 780 atgtccaaaa tagccaggaagaagaaacga gctgtcatta tgatggtgac agtggtggct 840 ctctttgctg tgtgctgggcaccattccat gttgtccata tgatgattga atacagtaat 900 tttgaaaagg aatatgatgatgtcacaatc aagatgattt ttgctatcgt gcaaattatt 960 ggattttcca actccatctgtaatcccatt gtctatgcat ttatgaatga aaacttcaaa 1020 aaaaatgttt tgtctgcagtttgttattgc atagtaaata aaaccttctc tccagcacaa 1080 aggcatggaa attcaggaattacaatgatg cggaagaaag caaagttttc cctcagagag 1140 aatccagtgg aggaaaccaaaggagaagca ttcagtgatg gcaacattga agtcaaattg 1200 tgtgaacaga cagaggagaagaaaaagctc aaacgacatc ttgctctctt taggtctgaa 1260 ctggctgaga attctcctttagacagtggg cattaa 1296 40 431 PRT Homo sapiens 40 Met Gln Ala Leu AsnIle Thr Pro Glu Gln Phe Ser Arg Leu Leu Arg 1 5 10 15 Asp His Asn LeuThr Arg Glu Gln Phe Ile Ala Leu Tyr Arg Leu Arg 20 25 30 Pro Leu Val TyrThr Pro Glu Leu Pro Gly Arg Ala Lys Leu Ala Leu 35 40 45 Val Leu Thr GlyVal Leu Ile Phe Ala Leu Ala Leu Phe Gly Asn Ala 50 55 60 Leu Val Phe TyrVal Val Thr Arg Ser Lys Ala Met Arg Thr Val Thr 65 70 75 80 Asn Ile PheIle Cys Ser Leu Ala Leu Ser Asp Leu Leu Ile Thr Phe 85 90 95 Phe Cys IlePro Val Thr Met Leu Gln Asn Ile Ser Asp Asn Trp Leu 100 105 110 Gly GlyAla Phe Ile Cys Lys Met Val Pro Phe Val Gln Ser Thr Ala 115 120 125 ValVal Thr Glu Met Leu Thr Met Thr Cys Ile Ala Val Glu Arg His 130 135 140Gln Gly Leu Val His Pro Phe Lys Met Lys Trp Gln Tyr Thr Asn Arg 145 150155 160 Arg Ala Phe Thr Met Leu Gly Val Val Trp Leu Val Ala Val Ile Val165 170 175 Gly Ser Pro Met Trp His Val Gln Gln Leu Glu Ile Lys Tyr AspPhe 180 185 190 Leu Tyr Glu Lys Glu His Ile Cys Cys Leu Glu Glu Trp ThrSer Pro 195 200 205 Val His Gln Lys Ile Tyr Thr Thr Phe Ile Leu Val IleLeu Phe Leu 210 215 220 Leu Pro Leu Met Val Met Leu Ile Leu Tyr Ser LysIle Gly Tyr Glu 225 230 235 240 Leu Trp Ile Lys Lys Arg Val Gly Asp GlySer Val Leu Arg Thr Ile 245 250 255 His Gly Lys Glu Met Ser Lys Ile AlaArg Lys Lys Lys Arg Ala Val 260 265 270 Ile Met Met Val Thr Val Val AlaLeu Phe Ala Val Cys Trp Ala Pro 275 280 285 Phe His Val Val His Met MetIle Glu Tyr Ser Asn Phe Glu Lys Glu 290 295 300 Tyr Asp Asp Val Thr IleLys Met Ile Phe Ala Ile Val Gln Ile Ile 305 310 315 320 Gly Phe Ser AsnSer Ile Cys Asn Pro Ile Val Tyr Ala Phe Met Asn 325 330 335 Glu Asn PheLys Lys Asn Val Leu Ser Ala Val Cys Tyr Cys Ile Val 340 345 350 Asn LysThr Phe Ser Pro Ala Gln Arg His Gly Asn Ser Gly Ile Thr 355 360 365 MetMet Arg Lys Lys Ala Lys Phe Ser Leu Arg Glu Asn Pro Val Glu 370 375 380Glu Thr Lys Gly Glu Ala Phe Ser Asp Gly Asn Ile Glu Val Lys Leu 385 390395 400 Cys Glu Gln Thr Glu Glu Lys Lys Lys Leu Lys Arg His Leu Ala Leu405 410 415 Phe Arg Ser Glu Leu Ala Glu Asn Ser Pro Leu Asp Ser Gly His420 425 430 41 24 DNA Artificial Sequence Novel Sequence 41 ctgtgtacagcagttcgcag agtg 24 42 24 DNA Artificial Sequence Novel Sequence 42gagtgccagg cagagcaggt agac 24 43 31 DNA Artificial Sequence NovelSequence 43 cccgaattcc tgcttgctcc cagcttggcc c 31 44 32 DNA ArtificialSequence Novel Sequence 44 tgtggatcct gctgtcaaag gtcccattcc gg 32 45 20DNA Artificial Sequence Novel Sequence 45 tcacaatgct aggtgtggtc 20 46 22DNA Artificial Sequence Novel Sequence 46 tgcatagaca atgggattac ag 22 47511 DNA Homo sapiens 47 tcacaatgct aggtgtggtc tggctggtgg cagtcatcgtaggatcaccc atgtggcacg 60 tgcaacaact tgagatcaaa tatgacttcc tatatgaaaaggaacacatc tgctgcttag 120 aagagtggac cagccctgtg caccagaaga tctacaccaccttcatcctt gtcatcctct 180 tcctcctgcc tcttatggtg atgcttattc tgtacgtaaaattggttatg aactttggat 240 aaagaaaaga gttggggatg gttcagtgct tcgaactattcatggaaaag aaatgtccaa 300 aatagccagg aagaagaaac gagctgtcat tatgatggtgacagtggtgg ctctctttgc 360 tgtgtgctgg gcaccattcc atgttgtcca tatgatgattgaatacagta attttgaaaa 420 ggaatatgat gatgtcacaa tcaagatgat ttttgctatcgtgcaaatta ttggattttc 480 caactccatc tgtaatccca ttgtctatgc a 511 48 21DNA Artificial Sequence Novel Sequence 48 ctgcttagaa gagtggacca g 21 4922 DNA Artificial Sequence Novel Sequence 49 ctgtgcacca gaagatctac ac 2250 21 DNA Artificial Sequence Novel Sequence 50 caaggatgaa ggtggtgtag a21 51 23 DNA Artificial Sequence Novel Sequence 51 gtgtagatct tctggtgcacagg 23 52 21 DNA Artificial Sequence Novel Sequence 52 gcaatgcaggtcatagtgag c 21 53 27 DNA Artificial Sequence Novel Sequence 53tggagcatgg tgacgggaat gcagaag 27 54 27 DNA Artificial Sequence NovelSequence 54 gtgatgagca ggtcactgag cgccaag 27 55 23 DNA ArtificialSequence Novel Sequence 55 gcaatgcagg cgcttaacat tac 23 56 22 DNAArtificial Sequence Novel Sequence 56 ttgggttaca atctgaaggg ca 22 57 23DNA Artificial Sequence Novel Sequence 57 actccgtgtc cagcaggact ctg 2358 24 DNA Artificial Sequence Novel Sequence 58 tgcgtgttcc tggaccctcacgtg 24 59 29 DNA Artificial Sequence Novel Sequence 59 caggccttggattttaatgt cagggatgg 29 60 27 DNA Artificial Sequence Novel Sequence 60ggagagtcag ctctgaaaga attcagg 27 61 27 DNA Artificial Sequence NovelSequence 61 tgatgtgatg ccagatacta atagcac 27 62 27 DNA ArtificialSequence Novel Sequence 62 cctgattcat ttaggtgaga ttgagac 27 63 26 DNAArtificial Sequence Novel Sequence 63 cccaagcttc cccaggtgta tttgat 26 6426 DNA Artificial Sequence Novel Sequence 64 gttggatcca cataatgcattttctc 26 65 1080 DNA Homo sapiens 65 atgattctca actcttctac tgaagatggtattaaaagaa tccaagatga ttgtcccaaa 60 gctggaaggc ataattacat atttgtcatgattcctactt tatacagtat catctttgtg 120 gtgggaatat ttggaaacag cttggtggtgatagtcattt acttttatat gaagctgaag 180 actgtggcca gtgtttttct tttgaatttagcactggctg acttatgctt tttactgact 240 ttgccactat gggctgtcta cacagctatggaataccgct ggccctttgg caattaccta 300 tgtaagattg cttcagccag cgtcagtttcaacctgtacg ctagtgtgtt tctactcacg 360 tgtctcagca ttgatcgata cctggctattgttcacccaa tgaagtcccg ccttcgacgc 420 acaatgcttg tagccaaagt cacctgcatcatcatttggc tgctggcagg cttggccagt 480 ttgccagcta taatccatcg aaatgtatttttcattgaga acaccaatat tacagtttgt 540 gctttccatt atgagtccca aaattcaacccttccgatag ggctgggcct gaccaaaaat 600 atactgggtt tcctgtttcc ttttctgatcattcttacaa gttatactct tatttggaag 660 gccctaaaga aggcttatga aattcagaagaacaaaccaa gaaatgatga tatttttaag 720 ataattatgg caattgtgct tttctttttcttttcctgga ttccccacca aatattcact 780 tttctggatg tattgattca actaggcatcatacgtgact gtagaattgc agatattgtg 840 gacacggcca tgcctatcac catttgtatagcttatttta acaattgcct gaatcctctt 900 ttttatggct ttctggggaa aaaatttaaaagatattttc tccagcttct aaaatatatt 960 cccccaaaag ccaaatccca ctcaaacctttcaacaaaaa tgagcacgct ttcctaccgc 1020 ccctcagata atgtaagctc atccaccaagaagcctgcac catgttttga ggttgagtga 1080 66 359 PRT Homo sapiens 66 Met IleLeu Asn Ser Ser Thr Glu Asp Gly Ile Lys Arg Ile Gln Asp 1 5 10 15 AspCys Pro Lys Ala Gly Arg His Asn Tyr Ile Phe Val Met Ile Pro 20 25 30 ThrLeu Tyr Ser Ile Ile Phe Val Val Gly Ile Phe Gly Asn Ser Leu 35 40 45 ValVal Ile Val Ile Tyr Phe Tyr Met Lys Leu Lys Thr Val Ala Ser 50 55 60 ValPhe Leu Leu Asn Leu Ala Leu Ala Asp Leu Cys Phe Leu Leu Thr 65 70 75 80Leu Pro Leu Trp Ala Val Tyr Thr Ala Met Glu Tyr Arg Trp Pro Phe 85 90 95Gly Asn Tyr Leu Cys Lys Ile Ala Ser Ala Ser Val Ser Phe Asn Leu 100 105110 Tyr Ala Ser Val Phe Leu Leu Thr Cys Leu Ser Ile Asp Arg Tyr Leu 115120 125 Ala Ile Val His Pro Met Lys Ser Arg Leu Arg Arg Thr Met Leu Val130 135 140 Ala Lys Val Thr Cys Ile Ile Ile Trp Leu Leu Ala Gly Leu AlaSer 145 150 155 160 Leu Pro Ala Ile Ile His Arg Asn Val Phe Phe Ile GluAsn Thr Asn 165 170 175 Ile Thr Val Cys Ala Phe His Tyr Glu Ser Gln AsnSer Thr Leu Pro 180 185 190 Ile Gly Leu Gly Leu Thr Lys Asn Ile Leu GlyPhe Leu Phe Pro Phe 195 200 205 Leu Ile Ile Leu Thr Ser Tyr Thr Leu IleTrp Lys Ala Leu Lys Lys 210 215 220 Ala Tyr Glu Ile Gln Lys Asn Lys ProArg Asn Asp Asp Ile Phe Lys 225 230 235 240 Ile Ile Met Ala Ile Val LeuPhe Phe Phe Phe Ser Trp Ile Pro His 245 250 255 Gln Ile Phe Thr Phe LeuAsp Val Leu Ile Gln Leu Gly Ile Ile Arg 260 265 270 Asp Cys Arg Ile AlaAsp Ile Val Asp Thr Ala Met Pro Ile Thr Ile 275 280 285 Cys Ile Ala TyrPhe Asn Asn Cys Leu Asn Pro Leu Phe Tyr Gly Phe 290 295 300 Leu Gly LysLys Phe Lys Arg Tyr Phe Leu Gln Leu Leu Lys Tyr Ile 305 310 315 320 ProPro Lys Ala Lys Ser His Ser Asn Leu Ser Thr Lys Met Ser Thr 325 330 335Leu Ser Tyr Arg Pro Ser Asp Asn Val Ser Ser Ser Thr Lys Lys Pro 340 345350 Ala Pro Cys Phe Glu Val Glu 355 67 27 DNA Artificial Sequence NovelSequence 67 accatgggca gcccctggaa cggcagc 27 68 39 DNA ArtificialSequence Novel Sequence 68 agaaccacca ccagcaggac gcggacggtc tgccggtgg 3969 39 DNA Artificial Sequence Novel Sequence 69 gtccgcgtcc tgctggtggtggttctggca tttataatt 39 70 33 DNA Artificial Sequence Novel Sequence 70cctggatcct tatcccatcg tcttcacgtt agc 33 71 26 DNA Artificial SequenceNovel Sequence 71 ctggaattct cctgccagca tggtga 26 72 30 DNA ArtificialSequence Novel Sequence 72 gcaggatcct atattgcgtg ctctgtcccc 30 73 999DNA Homo sapiens 73 atggtgaact ccacccaccg tgggatgcac acttctctgcacctctggaa ccgcagcagt 60 tacagactgc acagcaatgc cagtgagtcc cttggaaaaggctactctga tggagggtgc 120 tacgagcaac tttttgtctc tcctgaggtg tttgtgactctgggtgtcat cagcttgttg 180 gagaatatct tagtgattgt ggcaatagcc aagaacaagaatctgcattc acccatgtac 240 tttttcatct gcagcttggc tgtggctgat atgctggtgagcgtttcaaa tggatcagaa 300 accattatca tcaccctatt aaacagtaca gatacggatgcacagagttt cacagtgaat 360 attgataatg tcattgactc ggtgatctgt agctccttgcttgcatccat ttgcagcctg 420 ctttcaattg cagtggacag gtactttact atcttctatgctctccagta ccataacatt 480 atgacagtta agcgggttgg gatcagcata agttgtatctgggcagcttg cacggtttca 540 ggcattttgt tcatcattta ctcagatagt agtgctgtcatcatctgcct catcaccatg 600 ttcttcacca tgctggctct catggcttct ctctatgtccacatgttcct gatggccagg 660 cttcacatta agaggattgc tgtcctcccc ggcactggtgccatccgcca aggtgccaat 720 atgaagggag cgattacctt gaccatcctg attggcgtctttgttgtctg ctgggcccca 780 ttcttcctcc acttaatatt ctacatctct tgtcctcagaatccatattg tgtgtgcttc 840 atgtctcact ttaacttgta tctcatactg atcatgtgtaattcaatcat cgatcctctg 900 atttatgcac tccggagtca agaactgagg aaaaccttcaaagagatcat ctgttgctat 960 cccctgggag gcctttgtga cttgtctagc agatattaa 99974 332 PRT Homo sapiens 74 Met Val Asn Ser Thr His Arg Gly Met His ThrSer Leu His Leu Trp 1 5 10 15 Asn Arg Ser Ser Tyr Arg Leu His Ser AsnAla Ser Glu Ser Leu Gly 20 25 30 Lys Gly Tyr Ser Asp Gly Gly Cys Tyr GluGln Leu Phe Val Ser Pro 35 40 45 Glu Val Phe Val Thr Leu Gly Val Ile SerLeu Leu Glu Asn Ile Leu 50 55 60 Val Ile Val Ala Ile Ala Lys Asn Lys AsnLeu His Ser Pro Met Tyr 65 70 75 80 Phe Phe Ile Cys Ser Leu Ala Val AlaAsp Met Leu Val Ser Val Ser 85 90 95 Asn Gly Ser Glu Thr Ile Ile Ile ThrLeu Leu Asn Ser Thr Asp Thr 100 105 110 Asp Ala Gln Ser Phe Thr Val AsnIle Asp Asn Val Ile Asp Ser Val 115 120 125 Ile Cys Ser Ser Leu Leu AlaSer Ile Cys Ser Leu Leu Ser Ile Ala 130 135 140 Val Asp Arg Tyr Phe ThrIle Phe Tyr Ala Leu Gln Tyr His Asn Ile 145 150 155 160 Met Thr Val LysArg Val Gly Ile Ser Ile Ser Cys Ile Trp Ala Ala 165 170 175 Cys Thr ValSer Gly Ile Leu Phe Ile Ile Tyr Ser Asp Ser Ser Ala 180 185 190 Val IleIle Cys Leu Ile Thr Met Phe Phe Thr Met Leu Ala Leu Met 195 200 205 AlaSer Leu Tyr Val His Met Phe Leu Met Ala Arg Leu His Ile Lys 210 215 220Arg Ile Ala Val Leu Pro Gly Thr Gly Ala Ile Arg Gln Gly Ala Asn 225 230235 240 Met Lys Gly Ala Ile Thr Leu Thr Ile Leu Ile Gly Val Phe Val Val245 250 255 Cys Trp Ala Pro Phe Phe Leu His Leu Ile Phe Tyr Ile Ser CysPro 260 265 270 Gln Asn Pro Tyr Cys Val Cys Phe Met Ser His Phe Asn LeuTyr Leu 275 280 285 Ile Leu Ile Met Cys Asn Ser Ile Ile Asp Pro Leu IleTyr Ala Leu 290 295 300 Arg Ser Gln Glu Leu Arg Lys Thr Phe Lys Glu IleIle Cys Cys Tyr 305 310 315 320 Pro Leu Gly Gly Leu Cys Asp Leu Ser SerArg Tyr 325 330 75 32 DNA Artificial Sequence Novel Sequence 75ccgaagcttc gagctgagta aggcggcggg ct 32 76 31 DNA Artificial SequenceNovel Sequence 76 gtggaattca tttgccctgc ctcaaccccc a 31 77 1344 DNA Homosapiens 77 atggagctgc taaagctgaa ccggagcgtg cagggaaccg gacccgggccgggggcttcc 60 ctgtgccgcc cgggggcgcc tctcctcaac agcagcagtg tgggcaacctcagctgcgag 120 ccccctcgca ttcgcggagc cgggacacga gaattggagc tggccattagaatcactctt 180 tacgcagtga tcttcctgat gagcgttgga ggaaatatgc tcatcatcgtggtcctggga 240 ctgagccgcc gcctgaggac tgtcaccaat gccttcctcc tctcactggcagtcagcgac 300 ctcctgctgg ctgtggcttg catgcccttc accctcctgc ccaatctcatgggcacattc 360 atctttggca ccgtcatctg caaggcggtt tcctacctca tgggggtgtctgtgagtgtg 420 tccacgctaa gcctcgtggc catcgcactg gagcgatata gcgccatctgccgaccactg 480 caggcacgag tgtggcagac gcgctcccac gcggctcgcg tgattgtagccacgtggctg 540 ctgtccggac tactcatggt gccctacccc gtgtacactg tcgtgcaaccagtggggcct 600 cgtgtgctgc agtgcgtgca tcgctggccc agtgcgcggg tccgccagacctggtccgta 660 ctgctgcttc tgctcttgtt cttcatccca ggtgtggtta tggccgtggcctacgggctt 720 atctctcgcg agctctactt agggcttcgc tttgacggcg acagtgacagcgacagccaa 780 agcagggtcc gaaaccaagg cgggctgcca ggggctgttc accagaacgggcgttgccgg 840 cctgagactg gcgcggttgg caaagacagc gatggctgct acgtgcaacttccacgttcc 900 cggcctgccc tggagctgac ggcgctgacg gctcctgggc cgggatccggctcccggccc 960 acccaggcca agctgctggc taagaagcgc gtggtgcgaa tgttgctggtgatcgttgtg 1020 cttttttttc tgtgttggtt gccagtttat agtgccaaca cgtggcgcgcctttgatggc 1080 ccgggtgcac accgagcact ctcgggtgct cctatctcct tcattcacttgctgagctac 1140 gcctcggcct gtgtcaaccc cctggtctac tgcttcatgc accgtcgctttcgccaggcc 1200 tgcctggaaa cttgcgctcg ctgctgcccc cggcctccac gagctcgccccagggctctt 1260 cccgatgagg accctcccac tccctccatt gcttcgctgt ccaggcttagctacaccacc 1320 atcagcacac tgggccctgg ctga 1344 78 447 PRT Homo sapiens78 Met Glu Leu Leu Lys Leu Asn Arg Ser Val Gln Gly Thr Gly Pro Gly 1 510 15 Pro Gly Ala Ser Leu Cys Arg Pro Gly Ala Pro Leu Leu Asn Ser Ser 2025 30 Ser Val Gly Asn Leu Ser Cys Glu Pro Pro Arg Ile Arg Gly Ala Gly 3540 45 Thr Arg Glu Leu Glu Leu Ala Ile Arg Ile Thr Leu Tyr Ala Val Ile 5055 60 Phe Leu Met Ser Val Gly Gly Asn Met Leu Ile Ile Val Val Leu Gly 6570 75 80 Leu Ser Arg Arg Leu Arg Thr Val Thr Asn Ala Phe Leu Leu Ser Leu85 90 95 Ala Val Ser Asp Leu Leu Leu Ala Val Ala Cys Met Pro Phe Thr Leu100 105 110 Leu Pro Asn Leu Met Gly Thr Phe Ile Phe Gly Thr Val Ile CysLys 115 120 125 Ala Val Ser Tyr Leu Met Gly Val Ser Val Ser Val Ser ThrLeu Ser 130 135 140 Leu Val Ala Ile Ala Leu Glu Arg Tyr Ser Ala Ile CysArg Pro Leu 145 150 155 160 Gln Ala Arg Val Trp Gln Thr Arg Ser His AlaAla Arg Val Ile Val 165 170 175 Ala Thr Trp Leu Leu Ser Gly Leu Leu MetVal Pro Tyr Pro Val Tyr 180 185 190 Thr Val Val Gln Pro Val Gly Pro ArgVal Leu Gln Cys Val His Arg 195 200 205 Trp Pro Ser Ala Arg Val Arg GlnThr Trp Ser Val Leu Leu Leu Leu 210 215 220 Leu Leu Phe Phe Ile Pro GlyVal Val Met Ala Val Ala Tyr Gly Leu 225 230 235 240 Ile Ser Arg Glu LeuTyr Leu Gly Leu Arg Phe Asp Gly Asp Ser Asp 245 250 255 Ser Asp Ser GlnSer Arg Val Arg Asn Gln Gly Gly Leu Pro Gly Ala 260 265 270 Val His GlnAsn Gly Arg Cys Arg Pro Glu Thr Gly Ala Val Gly Lys 275 280 285 Asp SerAsp Gly Cys Tyr Val Gln Leu Pro Arg Ser Arg Pro Ala Leu 290 295 300 GluLeu Thr Ala Leu Thr Ala Pro Gly Pro Gly Ser Gly Ser Arg Pro 305 310 315320 Thr Gln Ala Lys Leu Leu Ala Lys Lys Arg Val Val Arg Met Leu Leu 325330 335 Val Ile Val Val Leu Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala340 345 350 Asn Thr Trp Arg Ala Phe Asp Gly Pro Gly Ala His Arg Ala LeuSer 355 360 365 Val Ala Pro Ile Ser Phe Ile His Leu Leu Ser Tyr Ala SerAla Cys 370 375 380 Val Asn Pro Leu Val Tyr Cys Phe Met His Arg Arg PheArg Gln Ala 385 390 395 400 Cys Leu Glu Thr Cys Ala Arg Cys Cys Pro ArgPro Pro Arg Ala Arg 405 410 415 Pro Arg Ala Leu Pro Asp Glu Asp Pro ProThr Pro Ser Ile Ala Ser 420 425 430 Leu Ser Arg Leu Ser Tyr Thr Thr IleSer Thr Leu Gly Pro Gly 435 440 445 79 30 DNA Artificial Sequence NovelSequence 79 tgcaagctta aaaaggaaaa aatgaacagc 30 80 30 DNA ArtificialSequence Novel Sequence 80 taaggatccc ttcccttcaa aacatccttg 30 81 1014DNA Homo sapiens 81 atgaacagca catgtattga agaacagcat gacctggatcactatttgtt tcccattgtt 60 tacatctttg tgattatagt cagcattcca gccaatattggatctctgtg tgtgtctttc 120 ctgcaaccca agaaggaaag tgaactagga atttacctcttcagtttgtc actatcagat 180 ttactctatg cattaactct ccctttatgg attgattatacttggaataa agacaactgg 240 actttctctc ctgccttgtg caaagggagt gcttttctcatgtacatgaa gttttacagc 300 agcacagcat tcctcacctg cattgccgtt gatcggtatttggctgttgt ctaccctttg 360 aagttttttt tcctaaggac aagaagaatt gcactcatggtcagcctgtc catctggata 420 ttggaaacca tcttcaatgc tgtcatgttg tgggaagatgaaacagttgt tgaatattgc 480 gatgccgaaa agtctaattt tactttatgc tatgacaaataccctttaga gaaatggcaa 540 atcaacctca acttgttcag gacgtgtaca ggctatgcaatacctttggt caccatcctg 600 atctgtaacc ggaaagtcta ccaagctgtg cggcacaataaagccacgga aaacaaggaa 660 aagaagagaa tcataaaact acttgtcagc atcacagttacttttgtctt atgctttact 720 ccctttcatg tgatgttgct gattcgctgc attttagagcatgctgtgaa cttcgaagac 780 cacagcaatt ctgggaagcg aacttacaca atgtatagaatcacggttgc attaacaagt 840 ttaaattgtg ttgctgatcc aattctgtac tgttttgttaccgaaacagg aagatatgat 900 atgtggaata tattaaaatt ctgcactggg aggtgtaatacatcacaaag acaaagaaaa 960 cgcatacttt ctgtgtctac aaaagatact atggaattagaggtccttga gtag 1014 82 337 PRT Homo sapiens 82 Met Asn Ser Thr Cys IleGlu Glu Gln His Asp Leu Asp His Tyr Leu 1 5 10 15 Phe Pro Ile Val TyrIle Phe Val Ile Ile Val Ser Ile Pro Ala Asn 20 25 30 Ile Gly Ser Leu CysVal Ser Phe Leu Gln Pro Lys Lys Glu Ser Glu 35 40 45 Leu Gly Ile Tyr LeuPhe Ser Leu Ser Leu Ser Asp Leu Leu Tyr Ala 50 55 60 Leu Thr Leu Pro LeuTrp Ile Asp Tyr Thr Trp Asn Lys Asp Asn Trp 65 70 75 80 Thr Phe Ser ProAla Leu Cys Lys Gly Ser Ala Phe Leu Met Tyr Met 85 90 95 Lys Phe Tyr SerSer Thr Ala Phe Leu Thr Cys Ile Ala Val Asp Arg 100 105 110 Tyr Leu AlaVal Val Tyr Pro Leu Lys Phe Phe Phe Leu Arg Thr Arg 115 120 125 Arg IleAla Leu Met Val Ser Leu Ser Ile Trp Ile Leu Glu Thr Ile 130 135 140 PheAsn Ala Val Met Leu Trp Glu Asp Glu Thr Val Val Glu Tyr Cys 145 150 155160 Asp Ala Glu Lys Ser Asn Phe Thr Leu Cys Tyr Asp Lys Tyr Pro Leu 165170 175 Glu Lys Trp Gln Ile Asn Leu Asn Leu Phe Arg Thr Cys Thr Gly Tyr180 185 190 Ala Ile Pro Leu Val Thr Ile Leu Ile Cys Asn Arg Lys Val TyrGln 195 200 205 Ala Val Arg His Asn Lys Ala Thr Glu Asn Lys Glu Lys LysArg Ile 210 215 220 Ile Lys Leu Leu Val Ser Ile Thr Val Thr Phe Val LeuCys Phe Thr 225 230 235 240 Pro Phe His Val Met Leu Leu Ile Arg Cys IleLeu Glu His Ala Val 245 250 255 Asn Phe Glu Asp His Ser Asn Ser Gly LysArg Thr Tyr Thr Met Tyr 260 265 270 Arg Ile Thr Val Ala Leu Thr Ser LeuAsn Cys Val Ala Asp Pro Ile 275 280 285 Leu Tyr Cys Phe Val Thr Glu ThrGly Arg Tyr Asp Met Trp Asn Ile 290 295 300 Leu Lys Phe Cys Thr Gly ArgCys Asn Thr Ser Gln Arg Gln Arg Lys 305 310 315 320 Arg Ile Leu Ser ValSer Thr Lys Asp Thr Met Glu Leu Glu Val Leu 325 330 335 Glu 83 40 DNAArtificial Sequence Novel Sequence 83 caggaagaag aaacgagctg tcattatgatggtgacagtg 40 84 40 DNA Artificial Sequence Novel Sequence 84 cactgtcaccatcataatga cagctcgttt cttcttcctg 40 85 30 DNA Artificial Sequence NovelSequence 85 ggccaccggc agaccaaacg cgtcctgctg 30 86 31 DNA ArtificialSequence Novel Sequence 86 ctccttcggt cctcctatcg ttgtcagaag t 31 87 37DNA Artificial Sequence Novel Sequence 87 ggaaaagaag agaatcaaaaaactacttgt cagcatc 37 88 31 DNA Artificial Sequence Novel Sequence 88ctccttcggt cctcctatcg ttgtcagaag t 31 89 1080 DNA Homo sapiens 89atgattctca actcttctac tgaagatggt attaaaagaa tccaagatga ttgtcccaaa 60gctggaaggc ataattacat atttgtcatg attcctactt tatacagtat catctttgtg 120gtgggaatat ttggaaacag cttggtggtg atagtcattt acttttatat gaagctgaag 180actgtggcca gtgtttttct tttgaattta gcactggctg acttatgctt tttactgact 240ttgccactat gggctgtcta cacagctatg gaataccgct ggccctttgg caattaccta 300tgtaagattg cttcagccag cgtcagtttc aacctgtacg ctagtgtgtt tctactcacg 360tgtctcagca ttgatcgata cctggctatt gttcacccaa tgaagtcccg ccttcgacgc 420acaatgcttg tagccaaagt cacctgcatc atcatttggc tgctggcagg cttggccagt 480ttgccagcta taatccatcg aaatgtattt ttcattgaga acaccaatat tacagtttgt 540gctttccatt atgagtccca aaattcaacc cttccgatag ggctgggcct gaccaaaaat 600atactgggtt tcctgtttcc ttttctgatc attcttacaa gttatactct tatttggaag 660gccctaaaga aggcttatga aattcagaag aacaaaccaa gaaatgatga tattaaaaag 720ataattatgg caattgtgct tttctttttc ttttcctgga ttccccacca aatattcact 780tttctggatg tattgattca actaggcatc atacgtgact gtagaattgc agatattgtg 840gacacggcca tgcctatcac catttgtata gcttatttta acaattgcct gaatcctctt 900ttttatggct ttctggggaa aaaatttaaa agatattttc tccagcttct aaaatatatt 960cccccaaaag ccaaatccca ctcaaacctt tcaacaaaaa tgagcacgct ttcctaccgc 1020ccctcagata atgtaagctc atccaccaag aagcctgcac catgttttga ggttgagtga 108090 359 PRT Homo sapiens 90 Met Ile Leu Asn Ser Ser Thr Glu Asp Gly IleLys Arg Ile Gln Asp 1 5 10 15 Asp Cys Pro Lys Ala Gly Arg His Asn TyrIle Phe Val Met Ile Pro 20 25 30 Thr Leu Tyr Ser Ile Ile Phe Val Val GlyIle Phe Gly Asn Ser Leu 35 40 45 Val Val Ile Val Ile Tyr Phe Tyr Met LysLeu Lys Thr Val Ala Ser 50 55 60 Val Phe Leu Leu Asn Leu Ala Leu Ala AspLeu Cys Phe Leu Leu Thr 65 70 75 80 Leu Pro Leu Trp Ala Val Tyr Thr AlaMet Glu Tyr Arg Trp Pro Phe 85 90 95 Gly Asn Tyr Leu Cys Lys Ile Ala SerAla Ser Val Ser Phe Asn Leu 100 105 110 Tyr Ala Ser Val Phe Leu Leu ThrCys Leu Ser Ile Asp Arg Tyr Leu 115 120 125 Ala Ile Val His Pro Met LysSer Arg Leu Arg Arg Thr Met Leu Val 130 135 140 Ala Lys Val Thr Cys IleIle Ile Trp Leu Leu Ala Gly Leu Ala Ser 145 150 155 160 Leu Pro Ala IleIle His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn 165 170 175 Ile Thr ValCys Ala Phe His Tyr Glu Ser Gln Asn Ser Thr Leu Pro 180 185 190 Ile GlyLeu Gly Leu Thr Lys Asn Ile Leu Gly Phe Leu Phe Pro Phe 195 200 205 LeuIle Ile Leu Thr Ser Tyr Thr Leu Ile Trp Lys Ala Leu Lys Lys 210 215 220Ala Tyr Glu Ile Gln Lys Asn Lys Pro Arg Asn Asp Asp Ile Lys Lys 225 230235 240 Ile Ile Met Ala Ile Val Leu Phe Phe Phe Phe Ser Trp Ile Pro His245 250 255 Gln Ile Phe Thr Phe Leu Asp Val Leu Ile Gln Leu Gly Ile IleArg 260 265 270 Asp Cys Arg Ile Ala Asp Ile Val Asp Thr Ala Met Pro IleThr Ile 275 280 285 Cys Ile Ala Tyr Phe Asn Asn Cys Leu Asn Pro Leu PheTyr Gly Phe 290 295 300 Leu Gly Lys Lys Phe Lys Arg Tyr Phe Leu Gln LeuLeu Lys Tyr Ile 305 310 315 320 Pro Pro Lys Ala Lys Ser His Ser Asn LeuSer Thr Lys Met Ser Thr 325 330 335 Leu Ser Tyr Arg Pro Ser Asp Asn ValSer Ser Ser Thr Lys Lys Pro 340 345 350 Ala Pro Cys Phe Glu Val Glu 35591 35 DNA Artificial Sequence Novel Sequence 91 ccaagaaatg atgatattaaaaagataatt atggc 35 92 31 DNA Artificial Sequence Novel Sequence 92ctccttcggt cctcctatcg ttgtcagaag t 31 93 1080 DNA Homo sapiens 93atgattctca actcttctac tgaagatggt attaaaagaa tccaagatga ttgtcccaaa 60gctggaaggc ataattacat atttgtcatg attcctactt tatacagtat catctttgtg 120gtgggaatat ttggaaacag cttggtggtg atagtcattt acttttatat gaagctgaag 180actgtggcca gtgtttttct tttgaattta gcactggctg acttatgctt tttactgact 240ttgccactat gggctgtcta cacagctatg gaataccgct ggccctttgg caattaccta 300tgtaagattg cttcagccag cgtcagtttc gccctgtacg ctagtgtgtt tctactcacg 360tgtctcagca ttgatcgata cctggctatt gttcacccaa tgaagtcccg ccttcgacgc 420acaatgcttg tagccaaagt cacctgcatc atcatttggc tgctggcagg cttggccagt 480ttgccagcta taatccatcg aaatgtattt ttcattgaga acaccaatat tacagtttgt 540gctttccatt atgagtccca aaattcaacc cttccgatag ggctgggcct gaccaaaaat 600atactgggtt tcctgtttcc ttttctgatc attcttacaa gttatactct tatttggaag 660gccctaaaga aggcttatga aattcagaag aacaaaccaa gaaatgatga tatttttaag 720ataattatgg caattgtgct tttctttttc ttttcctgga ttccccacca aatattcact 780tttctggatg tattgattca actaggcatc atacgtgact gtagaattgc agatattgtg 840gacacggcca tgcctatcac catttgtata gcttatttta acaattgcct gaatcctctt 900ttttatggct ttctggggaa aaaatttaaa agatattttc tccagcttct aaaatatatt 960cccccaaaag ccaaatccca ctcaaacctt tcaacaaaaa tgagcacgct ttcctaccgc 1020ccctcagata atgtaagctc atccaccaag aagcctgcac catgttttga ggttgagtga 108094 359 PRT Homo sapiens 94 Met Ile Leu Asn Ser Ser Thr Glu Asp Gly IleLys Arg Ile Gln Asp 1 5 10 15 Asp Cys Pro Lys Ala Gly Arg His Asn TyrIle Phe Val Met Ile Pro 20 25 30 Thr Leu Tyr Ser Ile Ile Phe Val Val GlyIle Phe Gly Asn Ser Leu 35 40 45 Val Val Ile Val Ile Tyr Phe Tyr Met LysLeu Lys Thr Val Ala Ser 50 55 60 Val Phe Leu Leu Asn Leu Ala Leu Ala AspLeu Cys Phe Leu Leu Thr 65 70 75 80 Leu Pro Leu Trp Ala Val Tyr Thr AlaMet Glu Tyr Arg Trp Pro Phe 85 90 95 Gly Asn Tyr Leu Cys Lys Ile Ala SerAla Ser Val Ser Phe Ala Leu 100 105 110 Tyr Ala Ser Val Phe Leu Leu ThrCys Leu Ser Ile Asp Arg Tyr Leu 115 120 125 Ala Ile Val His Pro Met LysSer Arg Leu Arg Arg Thr Met Leu Val 130 135 140 Ala Lys Val Thr Cys IleIle Ile Trp Leu Leu Ala Gly Leu Ala Ser 145 150 155 160 Leu Pro Ala IleIle His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn 165 170 175 Ile Thr ValCys Ala Phe His Tyr Glu Ser Gln Asn Ser Thr Leu Pro 180 185 190 Ile GlyLeu Gly Leu Thr Lys Asn Ile Leu Gly Phe Leu Phe Pro Phe 195 200 205 LeuIle Ile Leu Thr Ser Tyr Thr Leu Ile Trp Lys Ala Leu Lys Lys 210 215 220Ala Tyr Glu Ile Gln Lys Asn Lys Pro Arg Asn Asp Asp Ile Phe Lys 225 230235 240 Ile Ile Met Ala Ile Val Leu Phe Phe Phe Phe Ser Trp Ile Pro His245 250 255 Gln Ile Phe Thr Phe Leu Asp Val Leu Ile Gln Leu Gly Ile IleArg 260 265 270 Asp Cys Arg Ile Ala Asp Ile Val Asp Thr Ala Met Pro IleThr Ile 275 280 285 Cys Ile Ala Tyr Phe Asn Asn Cys Leu Asn Pro Leu PheTyr Gly Phe 290 295 300 Leu Gly Lys Lys Phe Lys Arg Tyr Phe Leu Gln LeuLeu Lys Tyr Ile 305 310 315 320 Pro Pro Lys Ala Lys Ser His Ser Asn LeuSer Thr Lys Met Ser Thr 325 330 335 Leu Ser Tyr Arg Pro Ser Asp Asn ValSer Ser Ser Thr Lys Lys Pro 340 345 350 Ala Pro Cys Phe Glu Val Glu 35595 26 DNA Artificial Sequence Novel Sequence 95 cccaagcttc cccaggtgtatttgat 26 96 29 DNA Artificial Sequence Novel Sequence 96 cctgcaggcgaaactgactc tggctgaag 29 97 42 DNA Artificial Sequence Novel Sequence 97ctgtacgcta gtgtgtttct actcacgtgt ctcagcattg at 42 98 26 DNA ArtificialSequence Novel Sequence 98 gttggatcca cataatgcat tttctc 26 99 1080 DNAHomo sapiens 99 atgattctca actcttctac tgaagatggt attaaaagaa tccaagatgattgtcccaaa 60 gctggaaggc ataattacat atttgtcatg attcctactt tatacagtatcatctttgtg 120 gtgggaatat ttggaaacag cttggtggtg atagtcattt acttttatatgaagctgaag 180 actgtggcca gtgtttttct tttgaattta gcactggctg acttatgctttttactgact 240 ttgccactat gggctgtcta cacagctatg gaataccgct ggccctttggcaattaccta 300 tgtaagattg cttcagccag cgtcagtttc aacctgtacg ctagtgtgtttctactcacg 360 tgtctcagca ttgatcgata cctggctatt gttcacccaa tgaagtcccgccttcgacgc 420 acaatgcttg tagccaaagt cacctgcatc atcatttggc tgctggcaggcttggccagt 480 ttgccagcta taatccatcg aaatgtattt ttcattgaga acaccaatattacagtttgt 540 gctttccatt atgagtccca aaattcaacc cttccgatag ggctgggcctgaccaaaaat 600 atactgggtt tcctgtttcc ttttctgatc attcttacaa gttattttggaattcgaaaa 660 cacttactga agacgaatag ctatgggaag aacaggataa cccgtgaccaagttaagaag 720 ataattatgg caattgtgct tttctttttc ttttcctgga ttccccaccaaatattcact 780 tttctggatg tattgattca actaggcatc atacgtgact gtagaattgcagatattgtg 840 gacacggcca tgcctatcac catttgtata gcttatttta acaattgcctgaatcctctt 900 ttttatggct ttctggggaa aaaatttaaa agatattttc tccagcttctaaaatatatt 960 cccccaaaag ccaaatccca ctcaaacctt tcaacaaaaa tgagcacgctttcctaccgc 1020 ccctcagata atgtaagctc atccaccaag aagcctgcac catgttttgaggttgagtga 1080 100 359 PRT Homo sapiens 100 Met Ile Leu Asn Ser Ser ThrGlu Asp Gly Ile Lys Arg Ile Gln Asp 1 5 10 15 Asp Cys Pro Lys Ala GlyArg His Asn Tyr Ile Phe Val Met Ile Pro 20 25 30 Thr Leu Tyr Ser Ile IlePhe Val Val Gly Ile Phe Gly Asn Ser Leu 35 40 45 Val Val Ile Val Ile TyrPhe Tyr Met Lys Leu Lys Thr Val Ala Ser 50 55 60 Val Phe Leu Leu Asn LeuAla Leu Ala Asp Leu Cys Phe Leu Leu Thr 65 70 75 80 Leu Pro Leu Trp AlaVal Tyr Thr Ala Met Glu Tyr Arg Trp Pro Phe 85 90 95 Gly Asn Tyr Leu CysLys Ile Ala Ser Ala Ser Val Ser Phe Asn Leu 100 105 110 Tyr Ala Ser ValPhe Leu Leu Thr Cys Leu Ser Ile Asp Arg Tyr Leu 115 120 125 Ala Ile ValHis Pro Met Lys Ser Arg Leu Arg Arg Thr Met Leu Val 130 135 140 Ala LysVal Thr Cys Ile Ile Ile Trp Leu Leu Ala Gly Leu Ala Ser 145 150 155 160Leu Pro Ala Ile Ile His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn 165 170175 Ile Thr Val Cys Ala Phe His Tyr Glu Ser Gln Asn Ser Thr Leu Pro 180185 190 Ile Gly Leu Gly Leu Thr Lys Asn Ile Leu Gly Phe Leu Phe Pro Phe195 200 205 Leu Ile Ile Leu Thr Ser Tyr Phe Gly Ile Arg Lys His Leu LeuLys 210 215 220 Thr Asn Ser Tyr Gly Lys Asn Arg Ile Thr Arg Asp Gln ValLys Lys 225 230 235 240 Ile Ile Met Ala Ile Val Leu Phe Phe Phe Phe SerTrp Ile Pro His 245 250 255 Gln Ile Phe Thr Phe Leu Asp Val Leu Ile GlnLeu Gly Ile Ile Arg 260 265 270 Asp Cys Arg Ile Ala Asp Ile Val Asp ThrAla Met Pro Ile Thr Ile 275 280 285 Cys Ile Ala Tyr Phe Asn Asn Cys LeuAsn Pro Leu Phe Tyr Gly Phe 290 295 300 Leu Gly Lys Lys Phe Lys Arg TyrPhe Leu Gln Leu Leu Lys Tyr Ile 305 310 315 320 Pro Pro Lys Ala Lys SerHis Ser Asn Leu Ser Thr Lys Met Ser Thr 325 330 335 Leu Ser Tyr Arg ProSer Asp Asn Val Ser Ser Ser Thr Lys Lys Pro 340 345 350 Ala Pro Cys PheGlu Val Glu 355 101 37 DNA Artificial Sequence Novel Sequence 101tccgaattcc aaaataactt gtaagaatga tcagaaa 37 102 33 DNA ArtificialSequence Novel Sequence 102 agatcttaag aagataatta tggcaattgt gct 33 10362 DNA Artificial Sequence Novel Sequence 103 aattcgaaaa cacttactgaagacgaatag ctatgggaag aacaggataa cccgtgacca 60 ag 62 104 62 DNAArtificial Sequence Novel Sequence 104 ttaacttggt cacgggttat cctgttcttcccatagctat tcgtcttcag taagtgtttt 60 cg 62 105 1083 DNA Homo sapiens 105atgattctca actcttctac tgaagatggt attaaaagaa tccaagatga ttgtcccaaa 60gctggaaggc ataattacat atttgtcatg attcctactt tatacagtat catctttgtg 120gtgggaatat ttggaaacag cttggtggtg atagtcattt acttttatat gaagctgaag 180actgtggcca gtgtttttct tttgaattta gcactggctg acttatgctt tttactgact 240ttgccactat gggctgtcta cacagctatg gaataccgct ggccctttgg caattaccta 300tgtaagattg cttcagccag cgtcagtttc aacctgtacg ctagtgtgtt tctactcacg 360tgtctcagca ttgatcgata cctggctatt gttcacccaa tgaagtcccg ccttcgacgc 420acaatgcttg tagccaaagt cacctgcatc atcatttggc tgctggcagg cttggccagt 480ttgccagcta taatccatcg aaatgtattt ttcattgaga acaccaatat tacagtttgt 540gctttccatt atgagtccca aaattcaacc cttccgatag ggctgggcct gaccaaaaat 600atactgggtt tcctgtttcc ttttctgatc attcttacaa gttatactct tatttggaag 660gccctaaaga aggcttatga aattcagaag aacaaaccaa gaaatgatga tatttttaag 720ataattatgg cagcaattgt gcttttcttt ttcttttcct ggattcccca ccaaatattc 780acttttctgg atgtattgat tcaactaggc atcatacgtg actgtagaat tgcagatatt 840gtggacacgg ccatgcctat caccatttgt atagcttatt ttaacaattg cctgaatcct 900cttttttatg gctttctggg gaaaaaattt aaaagatatt ttctccagct tctaaaatat 960attcccccaa aagccaaatc ccactcaaac ctttcaacaa aaatgagcac gctttcctac 1020cgcccctcag ataatgtaag ctcatccacc aagaagcctg caccatgttt tgaggttgag 1080tga 1083 106 360 PRT Homo sapiens 106 Met Ile Leu Asn Ser Ser Thr GluAsp Gly Ile Lys Arg Ile Gln Asp 1 5 10 15 Asp Cys Pro Lys Ala Gly ArgHis Asn Tyr Ile Phe Val Met Ile Pro 20 25 30 Thr Leu Tyr Ser Ile Ile PheVal Val Gly Ile Phe Gly Asn Ser Leu 35 40 45 Val Val Ile Val Ile Tyr PheTyr Met Lys Leu Lys Thr Val Ala Ser 50 55 60 Val Phe Leu Leu Asn Leu AlaLeu Ala Asp Leu Cys Phe Leu Leu Thr 65 70 75 80 Leu Pro Leu Trp Ala ValTyr Thr Ala Met Glu Tyr Arg Trp Pro Phe 85 90 95 Gly Asn Tyr Leu Cys LysIle Ala Ser Ala Ser Val Ser Phe Asn Leu 100 105 110 Tyr Ala Ser Val PheLeu Leu Thr Cys Leu Ser Ile Asp Arg Tyr Leu 115 120 125 Ala Ile Val HisPro Met Lys Ser Arg Leu Arg Arg Thr Met Leu Val 130 135 140 Ala Lys ValThr Cys Ile Ile Ile Trp Leu Leu Ala Gly Leu Ala Ser 145 150 155 160 LeuPro Ala Ile Ile His Arg Asn Val Phe Phe Ile Glu Asn Thr Asn 165 170 175Ile Thr Val Cys Ala Phe His Tyr Glu Ser Gln Asn Ser Thr Leu Pro 180 185190 Ile Gly Leu Gly Leu Thr Lys Asn Ile Leu Gly Phe Leu Phe Pro Phe 195200 205 Leu Ile Ile Leu Thr Ser Tyr Thr Leu Ile Trp Lys Ala Leu Lys Lys210 215 220 Ala Tyr Glu Ile Gln Lys Asn Lys Pro Arg Asn Asp Asp Ile PheLys 225 230 235 240 Ile Ile Met Ala Ala Ile Val Leu Phe Phe Phe Phe SerTrp Ile Pro 245 250 255 His Gln Ile Phe Thr Phe Leu Asp Val Leu Ile GlnLeu Gly Ile Ile 260 265 270 Arg Asp Cys Arg Ile Ala Asp Ile Val Asp ThrAla Met Pro Ile Thr 275 280 285 Ile Cys Ile Ala Tyr Phe Asn Asn Cys LeuAsn Pro Leu Phe Tyr Gly 290 295 300 Phe Leu Gly Lys Lys Phe Lys Arg TyrPhe Leu Gln Leu Leu Lys Tyr 305 310 315 320 Ile Pro Pro Lys Ala Lys SerHis Ser Asn Leu Ser Thr Lys Met Ser 325 330 335 Thr Leu Ser Tyr Arg ProSer Asp Asn Val Ser Ser Ser Thr Lys Lys 340 345 350 Pro Ala Pro Cys PheGlu Val Glu 355 360 107 26 DNA Artificial Sequence Novel Sequence 107cccaagcttc cccaggtgta tttgat 26 108 38 DNA Artificial Sequence NovelSequence 108 aagcacaatt gctgcataat tatcttaaaa atatcatc 38 109 39 DNAArtificial Sequence Novel Sequence 109 aagataatta tggcagcaat tgtgcttttctttttcttt 39 110 26 DNA Artificial Sequence Novel Sequence 110gttggatcca cataatgcat tttctc 26 111 1344 DNA Homo sapiens 111 atggagctgctaaagctgaa ccggagcgtg cagggaaccg gacccgggcc gggggcttcc 60 ctgtgccgcccgggggcgcc tctcctcaac agcagcagtg tgggcaacct cagctgcgag 120 ccccctcgcattcgcggagc cgggacacga gaattggagc tggccattag aatcactctt 180 tacgcagtgatcttcctgat gagcgttgga ggaaatatgc tcatcatcgt ggtcctggga 240 ctgagccgccgcctgaggac tgtcaccaat gccttcctcc tctcactggc agtcagcgac 300 ctcctgctggctgtggcttg catgcccttc accctcctgc ccaatctcat gggcacattc 360 atctttggcaccgtcatctg caaggcggtt tcctacctca tgggggtgtc tgtgagtgtg 420 tccacgctaagcctcgtggc catcgcactg gagcgatata gcgccatctg ccgaccactg 480 caggcacgagtgtggcagac gcgctcccac gcggctcgcg tgattgtagc cacgtggctg 540 ctgtccggactactcatggt gccctacccc gtgtacactg tcgtgcaacc agtggggcct 600 cgtgtgctgcagtgcgtgca tcgctggccc agtgcgcggg tccgccagac ctggtccgta 660 ctgctgcttctgctcttgtt cttcatccca ggtgtggtta tggccgtggc ctacgggctt 720 atctctcgcgagctctactt agggcttcgc tttgacggcg acagtgacag cgacagccaa 780 agcagggtccgaaaccaagg cgggctgcca ggggctgttc accagaacgg gcgttgccgg 840 cctgagactggcgcggttgg caaagacagc gatggctgct acgtgcaact tccacgttcc 900 cggcctgccctggagctgac ggcgctgacg gctcctgggc cgggatccgg ctcccggccc 960 acccaggccaagctgctggc taagaagcgc gtgaaacgaa tgttgctggt gatcgttgtg 1020 cttttttttctgtgttggtt gccagtttat agtgccaaca cgtggcgcgc ctttgatggc 1080 ccgggtgcacaccgagcact ctcgggtgct cctatctcct tcattcactt gctgagctac 1140 gcctcggcctgtgtcaaccc cctggtctac tgcttcatgc accgtcgctt tcgccaggcc 1200 tgcctggaaacttgcgctcg ctgctgcccc cggcctccac gagctcgccc cagggctctt 1260 cccgatgaggaccctcccac tccctccatt gcttcgctgt ccaggcttag ctacaccacc 1320 atcagcacactgggccctgg ctga 1344 112 447 PRT Homo sapiens 112 Met Glu Leu Leu LysLeu Asn Arg Ser Val Gln Gly Thr Gly Pro Gly 1 5 10 15 Pro Gly Ala SerLeu Cys Arg Pro Gly Ala Pro Leu Leu Asn Ser Ser 20 25 30 Ser Val Gly AsnLeu Ser Cys Glu Pro Pro Arg Ile Arg Gly Ala Gly 35 40 45 Thr Arg Glu LeuGlu Leu Ala Ile Arg Ile Thr Leu Tyr Ala Val Ile 50 55 60 Phe Leu Met SerVal Gly Gly Asn Met Leu Ile Ile Val Val Leu Gly 65 70 75 80 Leu Ser ArgArg Leu Arg Thr Val Thr Asn Ala Phe Leu Leu Ser Leu 85 90 95 Ala Val SerAsp Leu Leu Leu Ala Val Ala Cys Met Pro Phe Thr Leu 100 105 110 Leu ProAsn Leu Met Gly Thr Phe Ile Phe Gly Thr Val Ile Cys Lys 115 120 125 AlaVal Ser Tyr Leu Met Gly Val Ser Val Ser Val Ser Thr Leu Ser 130 135 140Leu Val Ala Ile Ala Leu Glu Arg Tyr Ser Ala Ile Cys Arg Pro Leu 145 150155 160 Gln Ala Arg Val Trp Gln Thr Arg Ser His Ala Ala Arg Val Ile Val165 170 175 Ala Thr Trp Leu Leu Ser Gly Leu Leu Met Val Pro Tyr Pro ValTyr 180 185 190 Thr Val Val Gln Pro Val Gly Pro Arg Val Leu Gln Cys ValHis Arg 195 200 205 Trp Pro Ser Ala Arg Val Arg Gln Thr Trp Ser Val LeuLeu Leu Leu 210 215 220 Leu Leu Phe Phe Ile Pro Gly Val Val Met Ala ValAla Tyr Gly Leu 225 230 235 240 Ile Ser Arg Glu Leu Tyr Leu Gly Leu ArgPhe Asp Gly Asp Ser Asp 245 250 255 Ser Asp Ser Gln Ser Arg Val Arg AsnGln Gly Gly Leu Pro Gly Ala 260 265 270 Val His Gln Asn Gly Arg Cys ArgPro Glu Thr Gly Ala Val Gly Lys 275 280 285 Asp Ser Asp Gly Cys Tyr ValGln Leu Pro Arg Ser Arg Pro Ala Leu 290 295 300 Glu Leu Thr Ala Leu ThrAla Pro Gly Pro Gly Ser Gly Ser Arg Pro 305 310 315 320 Thr Gln Ala LysLeu Leu Ala Lys Lys Arg Val Lys Arg Met Leu Leu 325 330 335 Val Ile ValVal Leu Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala 340 345 350 Asn ThrTrp Arg Ala Phe Asp Gly Pro Gly Ala His Arg Ala Leu Ser 355 360 365 ValAla Pro Ile Ser Phe Ile His Leu Leu Ser Tyr Ala Ser Ala Cys 370 375 380Val Asn Pro Leu Val Tyr Cys Phe Met His Arg Arg Phe Arg Gln Ala 385 390395 400 Cys Leu Glu Thr Cys Ala Arg Cys Cys Pro Arg Pro Pro Arg Ala Arg405 410 415 Pro Arg Ala Leu Pro Asp Glu Asp Pro Pro Thr Pro Ser Ile AlaSer 420 425 430 Leu Ser Arg Leu Ser Tyr Thr Thr Ile Ser Thr Leu Gly ProGly 435 440 445 113 34 DNA Artificial Sequence Novel Sequence 113cagcagcatg cgcttcacgc gcttcttagc ccag 34 114 35 DNA Artificial SequenceNovel Sequence 114 agaagcgcgt gaagcgcatg ctgctggtga tcgtt 35 115 33 DNAArtificial Sequence Novel Sequence 115 atggagaaaa gaatcaaaag aatgttctatata 33 116 33 DNA Artificial Sequence Novel Sequence 116 tatatagaacattcttttga ttcttttctc cat 33 117 30 DNA Artificial Sequence NovelSequence 117 cgctctctgg ccttgaagcg cacgctcagc 30 118 30 DNA ArtificialSequence Novel Sequence 118 gctgagcgtg cgcttcaagg ccagagagcg 30 119 30DNA Artificial Sequence Novel Sequence 119 cccaggaaaa aggtgaaagtcaaagttttc 30 120 30 DNA Artificial Sequence Novel Sequence 120gaaaactttg actttcacct ttttcctggg 30 121 27 DNA Artificial Sequence NovelSequence 121 ggggcgcggg tgaaacggct ggtgagc 27 122 27 DNA ArtificialSequence Novel Sequence 122 gctcaccagc cgtttcaccc gcgcccc 27 123 30 DNAArtificial Sequence Novel Sequence 123 ccccttgaaa agcctaagaa cttggtcatc30 124 30 DNA Artificial Sequence Novel Sequence 124 gatgaccaagttcttaggct tttcaagggg 30 125 32 DNA Artificial Sequence Novel Sequence125 gatctctaga atgaacagca catgtattga ag 32 126 35 DNA ArtificialSequence Novel Sequence 126 ctagggtacc cgctcaagga cctctaattc catag 35127 1296 DNA Homo sapiens 127 atgcaggcgc ttaacattac cccggagcagttctctcggc tgctgcggga ccacaacctg 60 acgcgggagc agttcatcgc tctgtaccggctgcgaccgc tcgtctacac cccagagctg 120 ccgggacgcg ccaagctggc cctcgtgctcaccggcgtgc tcatcttcgc cctggcgctc 180 tttggcaatg ctctggtgtt ctacgtggtgacccgcagca aggccatgcg caccgtcacc 240 aacatcttta tctgctcctt ggcgctcagtgacctgctca tcaccttctt ctgcattccc 300 gtcaccatgc tccagaacat ttccgacaactggctggggg gtgctttcat ttgcaagatg 360 gtgccatttg tccagtctac cgctgttgtgacagaaatgc tcactatgac ctgcattgct 420 gtggaaaggc accagggact tgtgcatccttttaaaatga agtggcaata caccaaccga 480 agggctttca caatgctagg tgtggtctggctggtggcag tcatcgtagg atcacccatg 540 tggcacgtgc aacaacttga gatcaaatatgacttcctat atgaaaagga acacatctgc 600 tgcttagaag agtggaccag ccctgtgcaccagaagatct acaccacctt catccttgtc 660 atcctcttcc tcctgcctct tatggtgatgcttattctgt acagtaaaat tggttatgaa 720 ctttggataa agaaaagagt tggggatggttcagtgcttc gaactattca tggaaaagaa 780 atgtccaaaa tagccaggaa gaagaaacgagctaagatta tgatggtgac agtggtggct 840 ctctttgctg tgtgctgggc accattccatgttgtccata tgatgattga atacagtaat 900 tttgaaaagg aatatgatga tgtcacaatcaagatgattt ttgctatcgt gcaaattatt 960 ggattttcca actccatctg taatcccattgtctatgcat ttatgaatga aaacttcaaa 1020 aaaaatgttt tgtctgcagt ttgttattgcatagtaaata aaaccttctc tccagcacaa 1080 aggcatggaa attcaggaat tacaatgatgcggaagaaag caaagttttc cctcagagag 1140 aatccagtgg aggaaaccaa aggagaagcattcagtgatg gcaacattga agtcaaattg 1200 tgtgaacaga cagaggagaa gaaaaagctcaaacgacatc ttgctctctt taggtctgaa 1260 ctggctgaga attctccttt agacagtgggcattaa 1296 128 431 PRT Homo sapiens 128 Met Gln Ala Leu Asn Ile Thr ProGlu Gln Phe Ser Arg Leu Leu Arg 1 5 10 15 Asp His Asn Leu Thr Arg GluGln Phe Ile Ala Leu Tyr Arg Leu Arg 20 25 30 Pro Leu Val Tyr Thr Pro GluLeu Pro Gly Arg Ala Lys Leu Ala Leu 35 40 45 Val Leu Thr Gly Val Leu IlePhe Ala Leu Ala Leu Phe Gly Asn Ala 50 55 60 Leu Val Phe Tyr Val Val ThrArg Ser Lys Ala Met Arg Thr Val Thr 65 70 75 80 Asn Ile Phe Ile Cys SerLeu Ala Leu Ser Asp Leu Leu Ile Thr Phe 85 90 95 Phe Cys Ile Pro Val ThrMet Leu Gln Asn Ile Ser Asp Asn Trp Leu 100 105 110 Gly Gly Ala Phe IleCys Lys Met Val Pro Phe Val Gln Ser Thr Ala 115 120 125 Val Val Thr GluMet Leu Thr Met Thr Cys Ile Ala Val Glu Arg His 130 135 140 Gln Gly LeuVal His Pro Phe Lys Met Lys Trp Gln Tyr Thr Asn Arg 145 150 155 160 ArgAla Phe Thr Met Leu Gly Val Val Trp Leu Val Ala Val Ile Val 165 170 175Gly Ser Pro Met Trp His Val Gln Gln Leu Glu Ile Lys Tyr Asp Phe 180 185190 Leu Tyr Glu Lys Glu His Ile Cys Cys Leu Glu Glu Trp Thr Ser Pro 195200 205 Val His Gln Lys Ile Tyr Thr Thr Phe Ile Leu Val Ile Leu Phe Leu210 215 220 Leu Pro Leu Met Val Met Leu Ile Leu Tyr Ser Lys Ile Gly TyrGlu 225 230 235 240 Leu Trp Ile Lys Lys Arg Val Gly Asp Gly Ser Val LeuArg Thr Ile 245 250 255 His Gly Lys Glu Met Ser Lys Ile Ala Arg Lys LysLys Arg Ala Lys 260 265 270 Ile Met Met Val Thr Val Val Ala Leu Phe AlaVal Cys Trp Ala Pro 275 280 285 Phe His Val Val His Met Met Ile Glu TyrSer Asn Phe Glu Lys Glu 290 295 300 Tyr Asp Asp Val Thr Ile Lys Met IlePhe Ala Ile Val Gln Ile Ile 305 310 315 320 Gly Phe Ser Asn Ser Ile CysAsn Pro Ile Val Tyr Ala Phe Met Asn 325 330 335 Glu Asn Phe Lys Lys AsnVal Leu Ser Ala Val Cys Tyr Cys Ile Val 340 345 350 Asn Lys Thr Phe SerPro Ala Gln Arg His Gly Asn Ser Gly Ile Thr 355 360 365 Met Met Arg LysLys Ala Lys Phe Ser Leu Arg Glu Asn Pro Val Glu 370 375 380 Glu Thr LysGly Glu Ala Phe Ser Asp Gly Asn Ile Glu Val Lys Leu 385 390 395 400 CysGlu Gln Thr Glu Glu Lys Lys Lys Leu Lys Arg His Leu Ala Leu 405 410 415Phe Arg Ser Glu Leu Ala Glu Asn Ser Pro Leu Asp Ser Gly His 420 425 430129 2040 DNA Homo sapiens 129 atgggcagcc cctggaacgg cagcgacggccccgaggggg cgcgggagcc gccgtggccc 60 gcgctgccgc cttgcgacga gcgccgctgctcgccctttc ccctgggggc gctggtgccg 120 gtgaccgctg tgtgcctgtg cctgttcgtcgtcggggtga gcggcaacgt ggtgaccgtg 180 atgctgatcg ggcgctaccg ggacatgcggaccaccacca acttgtacct gggcagcatg 240 gccgtgtccg acctactcat cctgctcgggctgccgttcg acctgtaccg cctctggcgc 300 tcgcggccct gggtgttcgg gccgctgctctgccgcctgt ccctctacgt gggcgagggc 360 tgcacctacg ccacgctgct gcacatgaccgcgctcagcg tcgagcgcta cctggccatc 420 tgccgcccgc tccgcgcccg cgtcttggtcacccggcgcc gcgtccgcgc gctcatcgct 480 gtgctctggg ccgtggcgct gctctctgccggtcccttct tgttcctggt gggcgtcgag 540 caggaccccg gcatctccgt agtcccgggcctcaatggca ccgcgcggat cgcctcctcg 600 cctctcgcct cgtcgccgcc tctctggctctcgcgggcgc caccgccgtc cccgccgtcg 660 gggcccgaga ccgcggaggc cgcggcgctgttcagccgcg aatgccggcc gagccccgcg 720 cagctgggcg cgctgcgtgt catgctgtgggtcaccaccg cctacttctt cctgcccttt 780 ctgtgcctca gcatcctcta cgggctcatcgggcgggagc tgtggagcag ccggcggccg 840 ctgcgaggcc cggccgcctc ggggcgggagagaggccacc ggcagaccaa acgcgtcctg 900 cgtaagtgga gccgccgtgg ttccaaagacgcctgcctgc agtccgcccc gccggggacc 960 gcgcaaacgc tgggtcccct tcccctgctcgcccagctct gggcgccgct tccagctccc 1020 tttcctattt cgattccagc ctccacccgccggtacttcc catcccccga gaaaaccatg 1080 tcctgtcccc caggagctct gggggaccccagggcgcttt gagggtggga tccccggatc 1140 cgattcagta accagcagtg cttttccagagcctctgaga ccagaaagga gagttggtaa 1200 ttcttaatcc aaccacctgt tagatgccacaaatgaggag tcctcacagt gctcttgaga 1260 agacgaggga gatttcatta agctaaaattttttatttaa tgttaagtga tgctgaaggc 1320 taaagtaaac cttgctcgta tcaaaaagtaaagattgtgc agacctgttg tagaattctt 1380 ttcaacagag aacagaaaac ttgtctccgaagtgggtttg tggaaggaag cctgccaagg 1440 cggcttgttc agagaaattg ctccttctggtttatgtcca gccttgataa cacatatggg 1500 agcctactat gcagttttaa agcaagtatccatgcagcct gcagcctggt cattttttct 1560 ggggtgagga tctgcctagg tagaagttttctctaattta ttttgctgtt acttgttatt 1620 gcagatggtt ccttgtcggg gtggggggtttatttgcttc ccaatgcttt tgttaatccc 1680 ggtgctgtgt cttatgttgc agtggtggtggttctggcat ttataatttg ctggttgccc 1740 ttccacgttg gcagaatcat ttacataaacacggaagatt cgcggatgat gtacttctct 1800 cagtacttta acatcgtcgc tctgcaacttttctatctga gcgcatctat caacccaatc 1860 ctctacaacc tcatttcaaa gaagtacagagcggcggcct ttaaactgct gctcgcaagg 1920 aagtccaggc cgagaggctt ccacagaagcagggacactg cgggggaagt tgcaggggac 1980 actggaggag acacggtggg ctacaccgagacaagcgcta acgtgaagac gatgggataa 2040 130 412 PRT Homo sapiens 130 MetGly Ser Pro Trp Asn Gly Ser Asp Gly Pro Glu Gly Ala Arg Glu 1 5 10 15Pro Pro Trp Pro Ala Leu Pro Pro Cys Asp Glu Arg Arg Cys Ser Pro 20 25 30Phe Pro Leu Gly Ala Leu Val Pro Val Thr Ala Val Cys Leu Cys Leu 35 40 45Phe Val Val Gly Val Ser Gly Asn Val Val Thr Val Met Leu Ile Gly 50 55 60Arg Tyr Arg Asp Met Arg Thr Thr Thr Asn Leu Tyr Leu Gly Ser Met 65 70 7580 Ala Val Ser Asp Leu Leu Ile Leu Leu Gly Leu Pro Phe Asp Leu Tyr 85 9095 Arg Leu Trp Arg Ser Arg Pro Trp Val Phe Gly Pro Leu Leu Cys Arg 100105 110 Leu Ser Leu Tyr Val Gly Glu Gly Cys Thr Tyr Ala Thr Leu Leu His115 120 125 Met Thr Ala Leu Ser Val Glu Arg Tyr Leu Ala Ile Cys Arg ProLeu 130 135 140 Arg Ala Arg Val Leu Val Thr Arg Arg Arg Val Arg Ala LeuIle Ala 145 150 155 160 Val Leu Trp Ala Val Ala Leu Leu Ser Ala Gly ProPhe Leu Phe Leu 165 170 175 Val Gly Val Glu Gln Asp Pro Gly Ile Ser ValVal Pro Gly Leu Asn 180 185 190 Gly Thr Ala Arg Ile Ala Ser Ser Pro LeuAla Ser Ser Pro Pro Leu 195 200 205 Trp Leu Ser Arg Ala Pro Pro Pro SerPro Pro Ser Gly Pro Glu Thr 210 215 220 Ala Glu Ala Ala Ala Leu Phe SerArg Glu Cys Arg Pro Ser Pro Ala 225 230 235 240 Gln Leu Gly Ala Leu ArgVal Met Leu Trp Val Thr Thr Ala Tyr Phe 245 250 255 Phe Leu Pro Phe LeuCys Leu Ser Ile Leu Tyr Gly Leu Ile Gly Arg 260 265 270 Glu Leu Trp SerSer Arg Arg Pro Leu Arg Gly Pro Ala Ala Ser Gly 275 280 285 Arg Glu ArgGly His Arg Gln Thr Lys Arg Val Leu Leu Val Val Val 290 295 300 Leu AlaPhe Ile Ile Cys Trp Leu Pro Phe His Val Gly Arg Ile Ile 305 310 315 320Tyr Ile Asn Thr Glu Asp Ser Arg Met Met Tyr Phe Ser Gln Tyr Phe 325 330335 Asn Ile Val Ala Leu Gln Leu Phe Tyr Leu Ser Ala Ser Ile Asn Pro 340345 350 Ile Leu Tyr Asn Leu Ile Ser Lys Lys Tyr Arg Ala Ala Ala Phe Lys355 360 365 Leu Leu Leu Ala Arg Lys Ser Arg Pro Arg Gly Phe His Arg SerArg 370 375 380 Asp Thr Ala Gly Glu Val Ala Gly Asp Thr Gly Gly Asp ThrVal Gly 385 390 395 400 Tyr Thr Glu Thr Ser Ala Asn Val Lys Thr Met Gly405 410 131 1344 DNA Homo sapiens 131 atggagctgc taaagctgaa ccggagcgtgcagggaaccg gacccgggcc gggggcttcc 60 ctgtgccgcc cgggggcgcc tctcctcaacagcagcagtg tgggcaacct cagctgcgag 120 ccccctcgca ttcgcggagc cgggacacgagaattggagc tggccattag aatcactctt 180 tacgcagtga tcttcctgat gagcgttggaggaaatatgc tcatcatcgt ggtcctggga 240 ctgagccgcc gcctgaggac tgtcaccaatgccttcctcc tctcactggc agtcagcgac 300 ctcctgctgg ctgtggcttg catgcccttcaccctcctgc ccaatctcat gggcacattc 360 atctttggca ccgtcatctg caaggcggtttcctacctca tgggggtgtc tgtgagtgtg 420 tccacgctaa gcctcgtggc catcgcactggagcgatata gcgccatctg ccgaccactg 480 caggcacgag tgtggcagac gcgctcccacgcggctcgcg tgattgtagc cacgtggctg 540 ctgtccggac tactcatggt gccctaccccgtgtacactg tcgtgcaacc agtggggcct 600 cgtgtgctgc agtgcgtgca tcgctggcccagtgcgcggg tccgccagac ctggtccgta 660 ctgctgcttc tgctcttgtt cttcatcccaggtgtggtta tggccgtggc ctacgggctt 720 atctctcgcg agctctactt agggcttcgctttgacggcg acagtgacag cgacagccaa 780 agcagggtcc gaaaccaagg cgggctgccaggggctgttc accagaacgg gcgttgccgg 840 cctgagactg gcgcggttgg caaagacagcgatggctgct acgtgcaact tccacgttcc 900 cggcctgccc tggagctgac ggcgctgacggctcctgggc cgggatccgg ctcccggccc 960 acccaggcca agctgctggc taagaagcgcgtgaaacgaa tgttgctggt gatcgttgtg 1020 cttttttttc tgtgttggtt gccagtttatagtgccaaca cgtggcgcgc ctttgatggc 1080 ccgggtgcac accgagcact ctcgggtgctcctatctcct tcattcactt gctgagctac 1140 gcctcggcct gtgtcaaccc cctggtctactgcttcatgc accgtcgctt tcgccaggcc 1200 tgcctggaaa cttgcgctcg ctgctgcccccggcctccac gagctcgccc cagggctctt 1260 cccgatgagg accctcccac tccctccattgcttcgctgt ccaggcttag ctacaccacc 1320 atcagcacac tgggccctgg ctga 1344132 447 PRT Homo sapiens 132 Met Glu Leu Leu Lys Leu Asn Arg Ser Val GlnGly Thr Gly Pro Gly 1 5 10 15 Pro Gly Ala Ser Leu Cys Arg Pro Gly AlaPro Leu Leu Asn Ser Ser 20 25 30 Ser Val Gly Asn Leu Ser Cys Glu Pro ProArg Ile Arg Gly Ala Gly 35 40 45 Thr Arg Glu Leu Glu Leu Ala Ile Arg IleThr Leu Tyr Ala Val Ile 50 55 60 Phe Leu Met Ser Val Gly Gly Asn Met LeuIle Ile Val Val Leu Gly 65 70 75 80 Leu Ser Arg Arg Leu Arg Thr Val ThrAsn Ala Phe Leu Leu Ser Leu 85 90 95 Ala Val Ser Asp Leu Leu Leu Ala ValAla Cys Met Pro Phe Thr Leu 100 105 110 Leu Pro Asn Leu Met Gly Thr PheIle Phe Gly Thr Val Ile Cys Lys 115 120 125 Ala Val Ser Tyr Leu Met GlyVal Ser Val Ser Val Ser Thr Leu Ser 130 135 140 Leu Val Ala Ile Ala LeuGlu Arg Tyr Ser Ala Ile Cys Arg Pro Leu 145 150 155 160 Gln Ala Arg ValTrp Gln Thr Arg Ser His Ala Ala Arg Val Ile Val 165 170 175 Ala Thr TrpLeu Leu Ser Gly Leu Leu Met Val Pro Tyr Pro Val Tyr 180 185 190 Thr ValVal Gln Pro Val Gly Pro Arg Val Leu Gln Cys Val His Arg 195 200 205 TrpPro Ser Ala Arg Val Arg Gln Thr Trp Ser Val Leu Leu Leu Leu 210 215 220Leu Leu Phe Phe Ile Pro Gly Val Val Met Ala Val Ala Tyr Gly Leu 225 230235 240 Ile Ser Arg Glu Leu Tyr Leu Gly Leu Arg Phe Asp Gly Asp Ser Asp245 250 255 Ser Asp Ser Gln Ser Arg Val Arg Asn Gln Gly Gly Leu Pro GlyAla 260 265 270 Val His Gln Asn Gly Arg Cys Arg Pro Glu Thr Gly Ala ValGly Lys 275 280 285 Asp Ser Asp Gly Cys Tyr Val Gln Leu Pro Arg Ser ArgPro Ala Leu 290 295 300 Glu Leu Thr Ala Leu Thr Ala Pro Gly Pro Gly SerGly Ser Arg Pro 305 310 315 320 Thr Gln Ala Lys Leu Leu Ala Lys Lys ArgVal Lys Arg Met Leu Leu 325 330 335 Val Ile Val Val Leu Phe Phe Leu CysTrp Leu Pro Val Tyr Ser Ala 340 345 350 Asn Thr Trp Arg Ala Phe Asp GlyPro Gly Ala His Arg Ala Leu Ser 355 360 365 Val Ala Pro Ile Ser Phe IleHis Leu Leu Ser Tyr Ala Ser Ala Cys 370 375 380 Val Asn Pro Leu Val TyrCys Phe Met His Arg Arg Phe Arg Gln Ala 385 390 395 400 Cys Leu Glu ThrCys Ala Arg Cys Cys Pro Arg Pro Pro Arg Ala Arg 405 410 415 Pro Arg AlaLeu Pro Asp Glu Asp Pro Pro Thr Pro Ser Ile Ala Ser 420 425 430 Leu SerArg Leu Ser Tyr Thr Thr Ile Ser Thr Leu Gly Pro Gly 435 440 445 133 1014DNA Homo sapiens 133 atgaacagca catgtattga agaacagcat gacctggatcactatttgtt tcccattgtt 60 tacatctttg tgattatagt cagcattcca gccaatattggatctctgtg tgtgtctttc 120 ctgcaagcaa agaaggaaag tgaactagga atttacctcttcagtttgtc actatcagat 180 ttactctatg cattaactct ccctttatgg attgattatacttggaataa agacaactgg 240 actttctctc ctgccttgtg caaagggagt gcttttctcatgtacatgaa tttttacagc 300 agcacagcat tcctcacctg cattgccgtt gatcggtatttggctgttgt ctaccctttg 360 aagttttttt tcctaaggac aagaagattt gcactcatggtcagcctgtc catctggata 420 ttggaaacca tcttcaatgc tgtcatgttg tgggaagatgaaacagttgt tgaatattgc 480 gatgccgaaa agtctaattt tactttatgc tatgacaaataccctttaga gaaatggcaa 540 atcaacctca acttgttcag gacgtgtaca ggctatgcaatacctttggt caccatcctg 600 atctgtaacc ggaaagtcta ccaagctgtg cggcacaataaagccacgga aaacaaggaa 660 aagaagagaa tcaaaaaact acttgtcagc atcacagttacttttgtctt atgctttact 720 ccctttcatg tgatgttgct gattcgctgc attttagagcatgctgtgaa cttcgaagac 780 cacagcaatt ctgggaagcg aacttacaca atgtatagaatcacggttgc attaacaagt 840 ttaaattgtg ttgctgatcc aattctgtac tgttttgttaccgaaacagg aagatatgat 900 atgtggaata tattaaaatt ctgcactggg aggtgtaatacatcacaaag acaaagaaaa 960 cgcatacttt ctgtgtctac aaaagatact atggaattagaggtccttga gtag 1014 134 337 PRT Homo sapiens 134 Met Asn Ser Thr CysIle Glu Glu Gln His Asp Leu Asp His Tyr Leu 1 5 10 15 Phe Pro Ile ValTyr Ile Phe Val Ile Ile Val Ser Ile Pro Ala Asn 20 25 30 Ile Gly Ser LeuCys Val Ser Phe Leu Gln Ala Lys Lys Glu Ser Glu 35 40 45 Leu Gly Ile TyrLeu Phe Ser Leu Ser Leu Ser Asp Leu Leu Tyr Ala 50 55 60 Leu Thr Leu ProLeu Trp Ile Asp Tyr Thr Trp Asn Lys Asp Asn Trp 65 70 75 80 Thr Phe SerPro Ala Leu Cys Lys Gly Ser Ala Phe Leu Met Tyr Met 85 90 95 Asn Phe TyrSer Ser Thr Ala Phe Leu Thr Cys Ile Ala Val Asp Arg 100 105 110 Tyr LeuAla Val Val Tyr Pro Leu Lys Phe Phe Phe Leu Arg Thr Arg 115 120 125 ArgPhe Ala Leu Met Val Ser Leu Ser Ile Trp Ile Leu Glu Thr Ile 130 135 140Phe Asn Ala Val Met Leu Trp Glu Asp Glu Thr Val Val Glu Tyr Cys 145 150155 160 Asp Ala Glu Lys Ser Asn Phe Thr Leu Cys Tyr Asp Lys Tyr Pro Leu165 170 175 Glu Lys Trp Gln Ile Asn Leu Asn Leu Phe Arg Thr Cys Thr GlyTyr 180 185 190 Ala Ile Pro Leu Val Thr Ile Leu Ile Cys Asn Arg Lys ValTyr Gln 195 200 205 Ala Val Arg His Asn Lys Ala Thr Glu Asn Lys Glu LysLys Arg Ile 210 215 220 Lys Lys Leu Leu Val Ser Ile Thr Val Thr Phe ValLeu Cys Phe Thr 225 230 235 240 Pro Phe His Val Met Leu Leu Ile Arg CysIle Leu Glu His Ala Val 245 250 255 Asn Phe Glu Asp His Ser Asn Ser GlyLys Arg Thr Tyr Thr Met Tyr 260 265 270 Arg Ile Thr Val Ala Leu Thr SerLeu Asn Cys Val Ala Asp Pro Ile 275 280 285 Leu Tyr Cys Phe Val Thr GluThr Gly Arg Tyr Asp Met Trp Asn Ile 290 295 300 Leu Lys Phe Cys Thr GlyArg Cys Asn Thr Ser Gln Arg Gln Arg Lys 305 310 315 320 Arg Ile Leu SerVal Ser Thr Lys Asp Thr Met Glu Leu Glu Val Leu 325 330 335 Glu 135 999DNA Homo sapiens 135 atggtgaact ccacccaccg tgggatgcac acttctctgcacctctggaa ccgcagcagt 60 tacagactgc acagcaatgc cagtgagtcc cttggaaaaggctactctga tggagggtgc 120 tacgagcaac tttttgtctc tcctgaggtg tttgtgactctgggtgtcat cagcttgttg 180 gagaatatct tagtgattgt ggcaatagcc aagaacaagaatctgcattc acccatgtac 240 tttttcatct gcagcttggc tgtggctgat atgctggtgagcgtttcaaa tggatcagaa 300 accattatca tcaccctatt aaacagtaca gatacggatgcacagagttt cacagtgaat 360 attgataatg tcattgactc ggtgatctgt agctccttgcttgcatccat ttgcagcctg 420 ctttcaattg cagtggacag gtactttact atcttctatgctctccagta ccataacatt 480 atgacagtta agcgggttgg gatcagcata agttgtatctgggcagcttg cacggtttca 540 ggcattttgt tcatcattta ctcagatagt agtgctgtcatcatctgcct catcaccatg 600 ttcttcacca tgctggctct catggcttct ctctatgtccacatgttcct gatggccagg 660 cttcacatta agaggattgc tgtcctcccc ggcactggtgccatccgcca aggtgccaat 720 atgaagggaa aaattacctt gaccatcctg attggcgtctttgttgtctg ctgggcccca 780 ttcttcctcc acttaatatt ctacatctct tgtcctcagaatccatattg tgtgtgcttc 840 atgtctcact ttaacttgta tctcatactg atcatgtgtaattcaatcat cgatcctctg 900 atttatgcac tccggagtca agaactgagg aaaaccttcaaagagatcat ctgttgctat 960 cccctgggag gcctttgtga cttgtctagc agatattaa 999136 332 PRT Homo sapiens 136 Met Val Asn Ser Thr His Arg Gly Met His ThrSer Leu His Leu Trp 1 5 10 15 Asn Arg Ser Ser Tyr Arg Leu His Ser AsnAla Ser Glu Ser Leu Gly 20 25 30 Lys Gly Tyr Ser Asp Gly Gly Cys Tyr GluGln Leu Phe Val Ser Pro 35 40 45 Glu Val Phe Val Thr Leu Gly Val Ile SerLeu Leu Glu Asn Ile Leu 50 55 60 Val Ile Val Ala Ile Ala Lys Asn Lys AsnLeu His Ser Pro Met Tyr 65 70 75 80 Phe Phe Ile Cys Ser Leu Ala Val AlaAsp Met Leu Val Ser Val Ser 85 90 95 Asn Gly Ser Glu Thr Ile Ile Ile ThrLeu Leu Asn Ser Thr Asp Thr 100 105 110 Asp Ala Gln Ser Phe Thr Val AsnIle Asp Asn Val Ile Asp Ser Val 115 120 125 Ile Cys Ser Ser Leu Leu AlaSer Ile Cys Ser Leu Leu Ser Ile Ala 130 135 140 Val Asp Arg Tyr Phe ThrIle Phe Tyr Ala Leu Gln Tyr His Asn Ile 145 150 155 160 Met Thr Val LysArg Val Gly Ile Ser Ile Ser Cys Ile Trp Ala Ala 165 170 175 Cys Thr ValSer Gly Ile Leu Phe Ile Ile Tyr Ser Asp Ser Ser Ala 180 185 190 Val IleIle Cys Leu Ile Thr Met Phe Phe Thr Met Leu Ala Leu Met 195 200 205 AlaSer Leu Tyr Val His Met Phe Leu Met Ala Arg Leu His Ile Lys 210 215 220Arg Ile Ala Val Leu Pro Gly Thr Gly Ala Ile Arg Gln Gly Ala Asn 225 230235 240 Met Lys Gly Lys Ile Thr Leu Thr Ile Leu Ile Gly Val Phe Val Val245 250 255 Cys Trp Ala Pro Phe Phe Leu His Leu Ile Phe Tyr Ile Ser CysPro 260 265 270 Gln Asn Pro Tyr Cys Val Cys Phe Met Ser His Phe Asn LeuTyr Leu 275 280 285 Ile Leu Ile Met Cys Asn Ser Ile Ile Asp Pro Leu IleTyr Ala Leu 290 295 300 Arg Ser Gln Glu Leu Arg Lys Thr Phe Lys Glu IleIle Cys Cys Tyr 305 310 315 320 Pro Leu Gly Gly Leu Cys Asp Leu Ser SerArg Tyr 325 330 137 33 DNA Artificial Sequence Novel Sequence 137gccaatatga agggaaaaat taccttgacc atc 33 138 31 DNA Artificial SequenceNovel Sequence 138 ctccttcggt cctcctatcg ttgtcagaag t 31 139 1842 DNAHomo sapiens 139 atggggccca ccctagcggt tcccaccccc tatggctgta ttggctgtaagctaccccag 60 ccagaatacc caccggctct aatcatcttt atgttctgcg cgatggttatcaccatcgtt 120 gtagacctaa tcggcaactc catggtcatt ttggctgtga cgaagaacaagaagctccgg 180 aattctggca acatcttcgt ggtcagtctc tctgtggccg atatgctggtggccatctac 240 ccataccctt tgatgctgca tgccatgtcc attgggggct gggatctgagccagttacag 300 tgccagatgg tcgggttcat cacagggctg agtgtggtcg gctccatcttcaacatcgtg 360 gcaatcgcta tcaaccgtta ctgctacatc tgccacagcc tccagtacgaacggatcttc 420 agtgtgcgca atacctgcat ctacctggtc atcacctgga tcatgaccgtcctggctgtc 480 ctgcccaaca tgtacattgg caccatcgag tacgatcctc gcacctacacctgcatcttc 540 aactatctga acaaccctgt cttcactgtt accatcgtct gcatccacttcgtcctccct 600 ctcctcatcg tgggtttctg ctacgtgagg atctggacca aagtgctggcggcccgtgac 660 cctgcagggc agaatcctga caaccaactt gctgaggttc gcaattttctaaccatgttt 720 gtgatcttcc tcctctttgc agtgtgctgg tgccctatca acgtgctcactgtcttggtg 780 gctgtcagtc cgaaggagat ggcaggcaag atccccaact ggctttatcttgcagcctac 840 ttcatagcct acttcaacag ctgcctcaac gctgtgatct acgggctcctcaatgagaat 900 ttccgaagag aatactggac catcttccat gctatgcggc accctatcatattcttccct 960 ggcctcatca gtgatattcg tgagatgcag gaggcccgta ccctggcccgcgcccgtgcc 1020 catgctcgcg accaagctcg tgaacaagac cgtgcccatg cctgtcctgctgtggaggaa 1080 accccgatga atgtccggaa tgttccatta cctggtgatg ctgcagctggccaccccgac 1140 cgtgcctctg gccaccctaa gccccattcc agatcctcct ctgcctatcgcaaatctgcc 1200 tctacccacc acaagtctgt ctttagccac tccaaggctg cctctggtcacctcaagcct 1260 gtctctggcc actccaagcc tgcctctggt caccccaagt ctgccactgtctaccctaag 1320 cctgcctctg tccatttcaa gggtgactct gtccatttca agggtgactctgtccatttc 1380 aagcctgact ctgttcattt caagcctgct tccagcaacc ccaagcccatcactggccac 1440 catgtctctg ctggcagcca ctccaagtct gccttcagtg ctgccaccagccaccctaaa 1500 cccatcaagc cagctaccag ccatgctgag cccaccactg ctgactatcccaagcctgcc 1560 actaccagcc accctaagcc cgctgctgct gacaaccctg agctctctgcctcccattgc 1620 cccgagatcc ctgccattgc ccaccctgtg tctgacgaca gtgacctccctgagtcggcc 1680 tctagccctg ccgctgggcc caccaagcct gctgccagcc agctggagtctgacaccatc 1740 gctgaccttc ctgaccctac tgtagtcact accagtacca atgattaccatgatgtcgtg 1800 gttgttgatg ttgaagatga tcctgatgaa atggctgtgt ga 1842 140613 PRT Homo sapiens 140 Met Gly Pro Thr Leu Ala Val Pro Thr Pro Tyr GlyCys Ile Gly Cys 1 5 10 15 Lys Leu Pro Gln Pro Glu Tyr Pro Pro Ala LeuIle Ile Phe Met Phe 20 25 30 Cys Ala Met Val Ile Thr Ile Val Val Asp LeuIle Gly Asn Ser Met 35 40 45 Val Ile Leu Ala Val Thr Lys Asn Lys Lys LeuArg Asn Ser Gly Asn 50 55 60 Ile Phe Val Val Ser Leu Ser Val Ala Asp MetLeu Val Ala Ile Tyr 65 70 75 80 Pro Tyr Pro Leu Met Leu His Ala Met SerIle Gly Gly Trp Asp Leu 85 90 95 Ser Gln Leu Gln Cys Gln Met Val Gly PheIle Thr Gly Leu Ser Val 100 105 110 Val Gly Ser Ile Phe Asn Ile Val AlaIle Ala Ile Asn Arg Tyr Cys 115 120 125 Tyr Ile Cys His Ser Leu Gln TyrGlu Arg Ile Phe Ser Val Arg Asn 130 135 140 Thr Cys Ile Tyr Leu Val IleThr Trp Ile Met Thr Val Leu Ala Val 145 150 155 160 Leu Pro Asn Met TyrIle Gly Thr Ile Glu Tyr Asp Pro Arg Thr Tyr 165 170 175 Thr Cys Ile PheAsn Tyr Leu Asn Asn Pro Val Phe Thr Val Thr Ile 180 185 190 Val Cys IleHis Phe Val Leu Pro Leu Leu Ile Val Gly Phe Cys Tyr 195 200 205 Val ArgIle Trp Thr Lys Val Leu Ala Ala Arg Asp Pro Ala Gly Gln 210 215 220 AsnPro Asp Asn Gln Leu Ala Glu Val Arg Asn Phe Leu Thr Met Phe 225 230 235240 Val Ile Phe Leu Leu Phe Ala Val Cys Trp Cys Pro Ile Asn Val Leu 245250 255 Thr Val Leu Val Ala Val Ser Pro Lys Glu Met Ala Gly Lys Ile Pro260 265 270 Asn Trp Leu Tyr Leu Ala Ala Tyr Phe Ile Ala Tyr Phe Asn SerCys 275 280 285 Leu Asn Ala Val Ile Tyr Gly Leu Leu Asn Glu Asn Phe ArgArg Glu 290 295 300 Tyr Trp Thr Ile Phe His Ala Met Arg His Pro Ile IlePhe Phe Pro 305 310 315 320 Gly Leu Ile Ser Asp Ile Arg Glu Met Gln GluAla Arg Thr Leu Ala 325 330 335 Arg Ala Arg Ala His Ala Arg Asp Gln AlaArg Glu Gln Asp Arg Ala 340 345 350 His Ala Cys Pro Ala Val Glu Glu ThrPro Met Asn Val Arg Asn Val 355 360 365 Pro Leu Pro Gly Asp Ala Ala AlaGly His Pro Asp Arg Ala Ser Gly 370 375 380 His Pro Lys Pro His Ser ArgSer Ser Ser Ala Tyr Arg Lys Ser Ala 385 390 395 400 Ser Thr His His LysSer Val Phe Ser His Ser Lys Ala Ala Ser Gly 405 410 415 His Leu Lys ProVal Ser Gly His Ser Lys Pro Ala Ser Gly His Pro 420 425 430 Lys Ser AlaThr Val Tyr Pro Lys Pro Ala Ser Val His Phe Lys Gly 435 440 445 Asp SerVal His Phe Lys Gly Asp Ser Val His Phe Lys Pro Asp Ser 450 455 460 ValHis Phe Lys Pro Ala Ser Ser Asn Pro Lys Pro Ile Thr Gly His 465 470 475480 His Val Ser Ala Gly Ser His Ser Lys Ser Ala Phe Ser Ala Ala Thr 485490 495 Ser His Pro Lys Pro Ile Lys Pro Ala Thr Ser His Ala Glu Pro Thr500 505 510 Thr Ala Asp Tyr Pro Lys Pro Ala Thr Thr Ser His Pro Lys ProAla 515 520 525 Ala Ala Asp Asn Pro Glu Leu Ser Ala Ser His Cys Pro GluIle Pro 530 535 540 Ala Ile Ala His Pro Val Ser Asp Asp Ser Asp Leu ProGlu Ser Ala 545 550 555 560 Ser Ser Pro Ala Ala Gly Pro Thr Lys Pro AlaAla Ser Gln Leu Glu 565 570 575 Ser Asp Thr Ile Ala Asp Leu Pro Asp ProThr Val Val Thr Thr Ser 580 585 590 Thr Asn Asp Tyr His Asp Val Val ValVal Asp Val Glu Asp Asp Pro 595 600 605 Asp Glu Met Ala Val 610 141 1842DNA Homo sapiens 141 atggggccca ccctagcggt tcccaccccc tatggctgtattggctgtaa gctaccccag 60 ccagaatacc caccggctct aatcatcttt atgttctgcgcgatggttat caccatcgtt 120 gtagacctaa tcggcaactc catggtcatt ttggctgtgacgaagaacaa gaagctccgg 180 aattctggca acatcttcgt ggtcagtctc tctgtggccgatatgctggt ggccatctac 240 ccataccctt tgatgctgca tgccatgtcc attgggggctgggatctgag ccagttacag 300 tgccagatgg tcgggttcat cacagggctg agtgtggtcggctccatctt caacatcgtg 360 gcaatcgcta tcaaccgtta ctgctacatc tgccacagcctccagtacga acggatcttc 420 agtgtgcgca atacctgcat ctacctggtc atcacctggatcatgaccgt cctggctgtc 480 ctgcccaaca tgtacattgg caccatcgag tacgatcctcgcacctacac ctgcatcttc 540 aactatctga acaaccctgt cttcactgtt accatcgtctgcatccactt cgtcctccct 600 ctcctcatcg tgggtttctg ctacgtgagg atctggaccaaagtgctggc ggcccgtgac 660 cctgcagggc agaatcctga caaccaactt gctgaggttcgcaataaact aaccatgttt 720 gtgatcttcc tcctctttgc agtgtgctgg tgccctatcaacgtgctcac tgtcttggtg 780 gctgtcagtc cgaaggagat ggcaggcaag atccccaactggctttatct tgcagcctac 840 ttcatagcct acttcaacag ctgcctcaac gctgtgatctacgggctcct caatgagaat 900 ttccgaagag aatactggac catcttccat gctatgcggcaccctatcat attcttctct 960 ggcctcatca gtgatattcg tgagatgcag gaggcccgtaccctggcccg cgcccgtgcc 1020 catgctcgcg accaagctcg tgaacaagac cgtgcccatgcctgtcctgc tgtggaggaa 1080 accccgatga atgtccggaa tgttccatta cctggtgatgctgcagctgg ccaccccgac 1140 cgtgcctctg gccaccctaa gccccattcc agatcctcctctgcctatcg caaatctgcc 1200 tctacccacc acaagtctgt ctttagccac tccaaggctgcctctggtca cctcaagcct 1260 gtctctggcc actccaagcc tgcctctggt caccccaagtctgccactgt ctaccctaag 1320 cctgcctctg tccatttcaa ggctgactct gtccatttcaagggtgactc tgtccatttc 1380 aagcctgact ctgttcattt caagcctgct tccagcaaccccaagcccat cactggccac 1440 catgtctctg ctggcagcca ctccaagtct gccttcaatgctgccaccag ccaccctaaa 1500 cccatcaagc cagctaccag ccatgctgag cccaccactgctgactatcc caagcctgcc 1560 actaccagcc accctaagcc cgctgctgct gacaaccctgagctctctgc ctcccattgc 1620 cccgagatcc ctgccattgc ccaccctgtg tctgacgacagtgacctccc tgagtcggcc 1680 tctagccctg ccgctgggcc caccaagcct gctgccagccagctggagtc tgacaccatc 1740 gctgaccttc ctgaccctac tgtagtcact accagtaccaatgattacca tgatgtcgtg 1800 gttgttgatg ttgaagatga tcctgatgaa atggctgtgtga 1842 142 613 PRT Homo sapiens 142 Met Gly Pro Thr Leu Ala Val Pro ThrPro Tyr Gly Cys Ile Gly Cys 1 5 10 15 Lys Leu Pro Gln Pro Glu Tyr ProPro Ala Leu Ile Ile Phe Met Phe 20 25 30 Cys Ala Met Val Ile Thr Ile ValVal Asp Leu Ile Gly Asn Ser Met 35 40 45 Val Ile Leu Ala Val Thr Lys AsnLys Lys Leu Arg Asn Ser Gly Asn 50 55 60 Ile Phe Val Val Ser Leu Ser ValAla Asp Met Leu Val Ala Ile Tyr 65 70 75 80 Pro Tyr Pro Leu Met Leu HisAla Met Ser Ile Gly Gly Trp Asp Leu 85 90 95 Ser Gln Leu Gln Cys Gln MetVal Gly Phe Ile Thr Gly Leu Ser Val 100 105 110 Val Gly Ser Ile Phe AsnIle Val Ala Ile Ala Ile Asn Arg Tyr Cys 115 120 125 Tyr Ile Cys His SerLeu Gln Tyr Glu Arg Ile Phe Ser Val Arg Asn 130 135 140 Thr Cys Ile TyrLeu Val Ile Thr Trp Ile Met Thr Val Leu Ala Val 145 150 155 160 Leu ProAsn Met Tyr Ile Gly Thr Ile Glu Tyr Asp Pro Arg Thr Tyr 165 170 175 ThrCys Ile Phe Asn Tyr Leu Asn Asn Pro Val Phe Thr Val Thr Ile 180 185 190Val Cys Ile His Phe Val Leu Pro Leu Leu Ile Val Gly Phe Cys Tyr 195 200205 Val Arg Ile Trp Thr Lys Val Leu Ala Ala Arg Asp Pro Ala Gly Gln 210215 220 Asn Pro Asp Asn Gln Leu Ala Glu Val Arg Asn Lys Leu Thr Met Phe225 230 235 240 Val Ile Phe Leu Leu Phe Ala Val Cys Trp Cys Pro Ile AsnVal Leu 245 250 255 Thr Val Leu Val Ala Val Ser Pro Lys Glu Met Ala GlyLys Ile Pro 260 265 270 Asn Trp Leu Tyr Leu Ala Ala Tyr Phe Ile Ala TyrPhe Asn Ser Cys 275 280 285 Leu Asn Ala Val Ile Tyr Gly Leu Leu Asn GluAsn Phe Arg Arg Glu 290 295 300 Tyr Trp Thr Ile Phe His Ala Met Arg HisPro Ile Ile Phe Phe Ser 305 310 315 320 Gly Leu Ile Ser Asp Ile Arg GluMet Gln Glu Ala Arg Thr Leu Ala 325 330 335 Arg Ala Arg Ala His Ala ArgAsp Gln Ala Arg Glu Gln Asp Arg Ala 340 345 350 His Ala Cys Pro Ala ValGlu Glu Thr Pro Met Asn Val Arg Asn Val 355 360 365 Pro Leu Pro Gly AspAla Ala Ala Gly His Pro Asp Arg Ala Ser Gly 370 375 380 His Pro Lys ProHis Ser Arg Ser Ser Ser Ala Tyr Arg Lys Ser Ala 385 390 395 400 Ser ThrHis His Lys Ser Val Phe Ser His Ser Lys Ala Ala Ser Gly 405 410 415 HisLeu Lys Pro Val Ser Gly His Ser Lys Pro Ala Ser Gly His Pro 420 425 430Lys Ser Ala Thr Val Tyr Pro Lys Pro Ala Ser Val His Phe Lys Ala 435 440445 Asp Ser Val His Phe Lys Gly Asp Ser Val His Phe Lys Pro Asp Ser 450455 460 Val His Phe Lys Pro Ala Ser Ser Asn Pro Lys Pro Ile Thr Gly His465 470 475 480 His Val Ser Ala Gly Ser His Ser Lys Ser Ala Phe Asn AlaAla Thr 485 490 495 Ser His Pro Lys Pro Ile Lys Pro Ala Thr Ser His AlaGlu Pro Thr 500 505 510 Thr Ala Asp Tyr Pro Lys Pro Ala Thr Thr Ser HisPro Lys Pro Ala 515 520 525 Ala Ala Asp Asn Pro Glu Leu Ser Ala Ser HisCys Pro Glu Ile Pro 530 535 540 Ala Ile Ala His Pro Val Ser Asp Asp SerAsp Leu Pro Glu Ser Ala 545 550 555 560 Ser Ser Pro Ala Ala Gly Pro ThrLys Pro Ala Ala Ser Gln Leu Glu 565 570 575 Ser Asp Thr Ile Ala Asp LeuPro Asp Pro Thr Val Val Thr Thr Ser 580 585 590 Thr Asn Asp Tyr His AspVal Val Val Val Asp Val Glu Asp Asp Pro 595 600 605 Asp Glu Met Ala Val610 143 33 DNA Artificial Sequence Novel Sequence 143 gctgaggttcgcaataaact aaccatgttt gtg 33 144 31 DNA Artificial Sequence NovelSequence 144 ctccttcggt cctcctatcg ttgtcagaag t 31 145 27 DNA ArtificialSequence Novel Sequence 145 ttagatatcg gggcccaccc tagcggt 27 146 29 DNAArtificial Sequence Novel Sequence 146 ggtaccccca cagccatttc atcaggatc29

What is claimed is:
 1. A cDNA encoding a non-endogenous, constitutivelyactivated version of a human G protein-coupled receptor comprisinghARE-3(F313K).
 2. A non-endogenous version of a human G protein-coupledreceptor encoded by the cDNA of claim
 1. 3. A Plasmid comprising aVector and the cDNA of claim
 1. 4. A Host Cell comprising the Plasmid ofclaim
 3. 5. A cDNA encoding a non-endogenous, constitutively activatedversion of a human G protein-coupled receptor comprising hARE-4(V233K)6. A non-endogenous version of a human G protein-coupled receptorencoded by the cDNA of claim
 5. 7. A Plasmid comprising a Vector and thecDNA of claim
 5. 8. A Host Cell comprising the Plasmid of claim
 7. 9. AcDNA encoding a non-endogenous, constitutively activated version of ahuman G protein-coupled receptor comprising hARE-5(A240K).
 10. Anon-endogenous version of a human G protein-coupled receptor encoded bythe cDNA of claim
 9. 11. A Plasmid comprising a Vector and the cDNA ofclaim
 5. 12. A Host Cell comprising the Plasmid of claim
 11. 13. A cDNAencoding a non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hGPCR14(L257K).
 14. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 13. 15. A Plasmid comprising a Vector and the cDNA of claim 13.16. A Host Cell comprising the Plasmid of claim
 15. 17. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hGPCR27(C283K).
 18. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 17. 19. A Plasmid comprising a Vector and the cDNA of claim 17.20. A Host Cell comprising the Plasmid of claim
 19. 21. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hARE-1(E232K).
 22. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 21. 23. A Plasmid comprising a Vector and the cDNA of claim 21.24. A Host Cell comprising the Plasmid of claim
 23. 25. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hARE-2(G285K).
 26. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 25. 27. A Plasmid comprising a Vector and the cDNA of claim 25.28. A Host Cell comprising the Plasmid of claim
 27. 29. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hPPR1(L239K).
 30. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 29. 31. A Plasmid comprising a Vector and the cDNA of claim 29.32. A Host Cell comprising the Plasmid of claim
 31. 33. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hG2A(K232A).
 34. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 33. 35. A Plasmid comprising a Vector and the cDNA of claim 33.36. A Host Cell comprising the Plasmid of claim
 35. 37. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hRUP3(L224K).
 38. A non-endogenousversion of a human G protein-coupled receptor encoded by the CDNA ofclaim
 37. 39. A Plasmid comprising a Vector and the cDNA of claim 37.40. A Host Cell comprising the Plasmid of claim
 39. 41. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hRUP5(A236K).
 42. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 41. 43. A Plasmid comprising a Vector and the cDNA of claim 41.44. A Host Cell comprising the Plasmid of claim
 42. 45. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hRUP6(N267K)
 46. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 45. 47. A Plasmid comprising a Vector and the CDNA of claim 45.48. A Host Cell comprising the Plasmid of claim
 47. 49. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hRUP7(A302K).
 50. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 49. 51. A Plasmid comprising a Vector and the CDNA of claim 49.52. A Host Cell comprising the Plasmid of claim
 51. 53. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hCHN4(V236K).
 54. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 53. 55. A Plasmid comprising a Vector and the cDNA of claim 53.56. A Host Cell comprising the Plasmid of claim
 55. 57. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hMC4(A244K).
 58. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 57. 59. A Plasmid comprising a Vector and the cDNA of claim 57.60. A Host Cell comprising the Plasmid of claim
 60. 61. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hCHN3(S284K).
 62. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 61. 63. A Plasmid comprising a Vector and the cDNA of claim 61.64. A Host Cell comprising the Plasmid of claim
 63. 65. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hCHN6(L352K).
 66. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 65. 67. A Plasmid comprising a Vector and the cDNA of claim 65.68. A Host Cell comprising the Plasmid of claim
 67. 69. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hCHN8(N235K).
 70. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 69. 71. A Plasmid comprising a Vector and the cDNA of claim 69.72. A Host Cell comprising the Plasmid of claim
 71. 73. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled receptor comprising hH9(F236K).
 74. A non-endogenousversion of a human G protein-coupled receptor encoded by the cDNA ofclaim
 73. 75. A Plasmid comprising a Vector and the cDNA of claim 73.76. A Host Cell comprising the Plasmid of claim
 74. 77. A cDNA encodinga non-endogenous, constitutively activated version of a human Gprotein-coupled AT1 receptor selected from the group consisting of:hAT1(F239K); hAT1(N111A); hAT1(AT2K255IC3); and hATI(A243+).
 78. Anon-endogenous version of a human G protein-coupled receptor encoded bya cDNA of claim
 77. 79. A Plasmid comprising a Vector and the cDNA ofclaim
 77. 80. A Host Cell comprising the Plasmid of claim 79.